The portal of Neviaser: a valid option for antegrade nailing of humerus fractures

Introduction: The objective of this retrospective non-randomized study was to evaluate the portal of Neviaser (PN) as an alternative approach in antegrade humeral nailing. Methods: The surgical approach for the straight antegrade intramedullary nail (SAIN) was either the anterolateral delta-split (group 2, n = 79) or the portal of Neviaser (group 3, n = 27). Length of surgery and time of radiation were extracted from charts. Patients stabilized using the PN were followed for a clinical and radiological exam. At follow-up we evaluated the DASH (Disability of the Arm, Shoulder and Hand) and CMS (Constant-Murley Score). Results: Between 10.2015 and 12.2018 191 proximal and diaphyseal humeral fractures were operated using either an angular stable extramedullary device (group 1, PHILOS®, n = 85) or a straight humeral nail (MultiLoc®, n = 106). Time of radiation and intervention followed a normal distribution. The mean length of surgery was 172.9 min (SD 91.5) in group 1, 121.5 min (SD 54.1) in group 2 and 96.4 min (SD 33.7) in group 3 (p < 0.01). Time of radiation was significantly different with 1.1 min (SD 0.6: group 1), 3.1 min (SD 1.6: group 2) and 2.9 min (SD 1.7: group 3) (p < 0.01). After a mean interval of 21.5 months (range 6–43 months) 14 / 27 patients of group 3 were available for a clinical and radiological follow-up. The mean DASH in group 3 was 25, the CMS reached 70. The age and sex weighted CMS mean value was 96%. Forward flexion was 131°, abduction 125°. The ratio of strength affected versus non-affected side was 4.4: 6.2 kg. Conclusions: The portal of Neviaser is a feasible and safe approach and is an alternative to the anterolateral delta-split. Length of surgery and time of radiation were significantly shorter. Level of evidence: IV

HIV-1 IN/Pol recruits LEDGF/p75 into viral particles

Background: The dynamic interaction between HIV and its host governs the replication of the virus and the study of the virus-host interplay is key to understand the viral lifecycle. The host factor lens epithelium-derived growth factor (LEDGF/p75) tethers the HIV preintegration complex to the chromatin through a direct interaction with integrase (IN). Small molecules that bind the LEDGF/p75 binding pocket of the HIV IN dimer (LEDGINs) block HIV replication through a multimodal mechanism impacting early and late stage replication including HIV maturation. Furthermore, LEDGF/p75 has been identified as a Pol interaction partner. This raised the question whether LEDGF/p75 besides acting as a molecular tether in the target cell, also affects late steps of HIV replication. Results: LEDGF/p75 is recruited into HIV-1 particles through direct interaction with the viral IN (or Pol polyprotein) and is a substrate for HIV-1 protease. Incubation in the presence of HIV-1 protease inhibitors resulted in detection of full-length LEDGF/p75 in purified viral particles. We also demonstrate that inhibition of LEDGF/p75-IN interaction by specific mutants or LEDGINs precludes incorporation of LEDGF/p75 in virions, underscoring the specificity of the uptake. LEDGF/p75 depletion did however not result in altered LEDGIN potency. Conclusion: Together, these results provide evidence for an IN/Pol mediated uptake of LEDGF/p75 in viral particles and a specific cleavage by HIV protease. Understanding of the possible role of LEDGF/p75 or its cleavage fragments in the viral particle awaits further experimentation

HIV integrase-host interactions as novel therapeutic targets: validation, characterisation and drug discovery

Replication of the human immunodeficiency virus type-1 (HIV-1) relies on the presence of cellular proteins or so-called cofactors to complete its replication cycle. Interactions between these cofactors and viral proteins are often essential and as such interesting targets for drug development. This work focuses on the validation and characterization of HIV-1 integrase (IN)-host interactions as novel therapeutic targets for drug discovery and the evaluation of small molecules targeting the interaction between HIV integrase and its cellular cofactor LEDGF/p75. In 2003, our group validated the cellular protein LEDGF/p75 as a cofactor of HIV-1 IN. LEDGF/p75 targets viral integration towards active chromatin and influences IN multimerization and catalytic activity. In 2010 our group reported on the discovery of LEDGINs, potent small molecules targeting the LEDGF/p75 binding pocket on HIV IN. LEDGINs are currently in early clinical development. LEDGINs displace LEDGF/p75 from HIV IN, preventing targeting to active chromatin, and allosterically inhibit HIV integrase activity. LEDGINs do not only block the integration step, but surprisingly also affect the infectivity of newly produced viral particles. To unravel the mechanism behind this late block, we investigated two hypotheses: 1.) The late activity of LEDGINs is mediated by their effect on IN (or Pol) multimerization 2.) LEDGINs prevent incorporation of LEDGF/p75 in viral particles. In chapter 3 we discuss the first hypothesis. We demonstrate that LEDGINs potently inhibit the late stages of HIV replication, with half maximal effective concentrations (EC50’s) in the nanomolar range. Furthermore, we show that addition of LEDGINs during virus production leads to defective viruses with a nucleoprotein complex outside the capsid cone, due to aberrant multimerization of HIV IN or its precursor protein Pol. In chapter 4 we investigate the second hypothesis. While LEDGF/p75 depletion in producer cells does not result in altered LEDGIN potency, we demonstrate that LEDGF/p75 is recruited into HIV-1 particles through a direct interaction with the viral IN (or the Pol precursor protein). We show that LEDGF/p75 can be processed by the viral protease in viral particles. The importance of LEDGF/p75 or processed fragments for viral replication is however not clear. In chapter 5 we validated the cellular protein YB-1, Y-box-binding protein 1, as a new cofactor of HIV replication. YB-1 depletion resulted into hampered HIV-1 replication in different cell lines. Our data revealed hampered virion production due to interference with the viral RNA metabolism. In addition, using quantitative qPCR and a microscopy based nuclear import test, we demonstrated that YB-1 knockdown leads to a block in nuclear import of HIV-1. We confirmed an interaction with HIV integrase using co-immunoprecipitation and Western blotting. Yet, a direct interaction using recombinant proteins could not be confirmed since YB-1 seemed to bind most proteins non-specifically. Together, our results indicate that YB-1 affects multiple stages of HIV replication. In conclusion, in the context of virus-host interactions we unraveled the mechanism of LEDGINs, showed the presence of LEDGF/p75 in viral particles and characterized YB-1 as a cofactor of HIV-1 replication.DANKWOORD I ABSTRACT III SAMENVATTING V LIST OF ABBREVIATIONS VII TABLE OF CONTENTS XIII 1 GENERAL INTRODUCTION 3 1.1 HIV AND AIDS 3 1.1.1 Epidemiology and classification 3 1.1.2 The HIV virus 3 1.1.3 The physiopathology of AIDS 5 1.2 CURRENT STRATEGIES AND CHALLENGES 7 1.2.1 Prevention of HIV-1 infection 7 1.2.2 An update on current treatment strategies 7 1.2.3 An HIV vaccine: challenges, setbacks and baby steps forward 11 1.2.4 The long road towards a cure 12 1.3 TARGETING COFACTORS OF HIV REPLICATION 15 1.3.1 An introduction to cofactors and restriction factors 21 1.4 TARGETING THE INTEGRASE-LEDGF/P75 INTERACTION 23 1.4.1 Integration of the viral DNA into the host cell chromatin 23 1.4.2 The role of LEDGF/p75 during HIV replication 24 1.4.3 Targeting the LEDGF/p75-integrase interaction 25 1.4.4 Mechanism of inhibitors targeting the LEDGF/p75 integrase pocket 29 1.5 CONCLUSION 30 2 OBJECTIVES AND DESIGN OF THE PROJECT 33 2.1 CHARACTERIZATION OF IN INHIBITORS TARGETING THE LEDGF/P75 BINDING SITE 33 2.1.1 The effect of LEDGINs on HIV-1 IN/Pol polyprotein multimerization 33 2.1.2 The role of LEDGF/p75 in late stage HIV-1 replication 33 2.2 VALIDATION OF YB-1 AS A NEW HIV-1 COFACTOR 34 2.3 DESIGN OF THE PROJECTS 34 2.3.1 Characterization of IN inhibitors targeting the LEDGF/p75 binding site 34 2.3.2 Validation of a new HIV cofactor 35 3 LEDGINS INHIBIT HIV LATE STAGE REPLICATION POTENTLY THROUGH STIMULATING IN/POL MULTIMERIZATION 39 3.1 ABSTRACT 39 3.2 INTRODUCTION 39 3.3 MATERIALS AND METHODS 40 3.3.1 Cells 40 3.3.2 Virus production and infectivity assays 40 3.3.3 Cloning of the Pol bacterial expression construct 41 3.3.4 Purification of recombinant proteins 41 3.3.5 AlphaScreen 41 3.3.6 Electron microscopy 42 3.4 RESULTS AND DISCUSSION 42 3.4.1 LEDGINs are potent inhibitors of HIV late stage replication 42 3.4.2 LEDGINs enhance HIV-1 IN/Pol polyprotein multimerization during virus production 43 3.5 ACKNOWLEDGMENTS 45 4 HIV-1 IN/POL RECRUITS LEDGF/P75 INTO VIRAL PARTICLES 49 4.1 ABSTRACT 49 4.2 INTRODUCTION 49 4.3 MATERIALS AND METHODS 50 4.3.1 Reagents 50 4.3.2 Cell culture and generation of cell lines 51 4.3.3 Virus production from LEDGF/p75 Knockout cells 52 4.3.4 Virus production by transfection 52 4.3.5 Infectivity assays 52 4.3.6 Purification of HIV particles 52 4.3.7 Ellman acetylcholine esterase activity assay 53 4.3.8 Semi-quantitative p24 ELISA 53 4.3.9 In vitro HIV-1 protease (PR) assay and detection of the cleavage products 53 4.3.10 Purification of recombinant proteins 54 4.3.11 AlphaScreen protein-protein interaction assay 54 4.3.12 Pull-down assays 55 4.3.13 Gel electrophoresis and immunoblot analysis for LEDGF/p75 uptake in virions. 55 4.3.14 Electron microscopy 55 4.4 RESULTS 56 4.4.1 LEDGF/p75 is incorporated in HIV-1 particles and is a substrate of HIV-1 protease 56 4.4.2 Identification of HIV-1 protease cleavage sites in LEDGF/p75 59 4.4.3 LEDGF/p75 is recruited into the viral particle through interaction with HIV integrase 61 4.4.4 LEDGF/p75 interacts with the HIV-Pol polyprotein 62 4.4.5 LEDGIN potency is independent of the presence of LEDGF/p75 in virus producer cells 64 4.5 DISCUSSION 65 4.6 CONCLUSION 67 4.7 ACKNOWLEDGMENTS 67 4.8 SUPPLEMENTARY INFORMATION 68 4.8.1 Supplementary tables 68 4.8.2 Supplementary Figures 71 5 Y-BOX-BINDING PROTEIN 1 SUPPORTS THE EARLY AND LATE STEPS OF HIV REPLICATION 77 5.1 ABSTRACT 77 5.2 INTRODUCTION 77 5.3 MATERIALS AND METHODS 79 5.3.1 Cell culture 79 5.3.2 Cloning 80 5.3.3 Vector production 80 5.3.4 Generation of stable cell lines 80 5.3.5 Western blotting and immunocytochemistry 81 5.3.6 Viral breakthrough experiments 81 5.3.7 Pre-Integration-Complex (PIC) assay 81 5.3.8 Analysis of infectivity/transduction efficiency 82 5.3.9 Analysis of HIV late effects 82 5.3.10 Quantitative PCR 83 5.3.11 Integration site sequencing 83 5.3.12 Transmission Electron Microscopy 84 5.3.13 Mass Spectrometry. 84 5.3.14 Co-immunoprecipitation 84 5.3.15 Protein purification and AlphaScreen binding assay. 85 5.4 RESULTS 85 5.4.1 YB-1 interacts with retroviral integrases 85 5.4.2 YB-1 supports HIV replication 88 5.4.3 Viral particle production is hampered by YB-1 depletion due to reduced viral RNA levels 90 5.4.4 YB-1 is important for the early steps of HIV replication 92 5.5 DISCUSSION 96 5.6 ACKNOWLEDGMENTS 98 5.7 SUPPLEMENTARY INFORMATION 99 5.7.1 Supplementary tables 99 5.7.2 Supplementary figures 101 6 CONCLUDING DISCUSSION 109 6.1 LEDGINS, COMPOUNDS WITH A DUAL MECHANISM, CAN INTERACT WITH THE HIV POL PRECURSOR PROTEIN IN VITRO 109 6.2 THE LATE EFFECT OF LEDGINS REVEALS THE PRESENCE OF LEDGF/P75 IN VIRAL PARTICLES 111 6.3 YB-1 AS A NEW COFACTOR OF HIV-1 REPLICATION 112 6.4 TARGETING HIV VIRUS-HOST INTERACTIONS, BRIDGES TO A BRIGHTER FUTURE? 114 REFERENCES 117 POPULAR SUMMARY 133 POPULAIRE SAMENVATTING 135 CURRICULUM VITAE 137nrpages: 162status: publishe

Convincing consumers to share personal data: double-edged effect of offering money

International audienceThe purpose of this paper is to demonstrate how offering control on data usage and offering money can increase willingness to share private information with a data broker. Design/methodology/approach Personal data are collected for internet users with a Web questionnaire. In an experimental framework, compensations control money are manipulated and consumers' data sharing is explained by sensitivity and regulatory focus. Findings Offering control increases willingness to disclose personal data, even sensitive one, but the effect is not moderated by regulatory focus. Offering monetary compensation has a negative, but small, effect on willingness to share personal data, and the effect is moderated by regulatory focus

Targeting virus-host interactions of HIV replication

Cellular proteins that are hijacked by HIV in order to complete its replication cycle, form attractive new targets for antiretroviral therapy. In particular, the protein-protein interactions between these cellular proteins (cofactors) and viral proteins are of great interest to develop new therapies. Research efforts have led to the validation of different cofactors and some successes in therapeutic applications. Maraviroc, the first cofactor inhibitor approved for human medicinal use, provided a proof of concept. Furthermore, compounds developed as Integrase-LEDGF/p75 interaction inhibitors (LEDGINs) have advanced to early clinical trials. Other compounds targeting cofactors and cofactor-viral protein interactions are currently under development. Likewise, interactions between cellular restriction factors and their counteracting HIV protein might serve as interesting targets in order to impair HIV replication. In this respect, compounds targeting the Vif-APOBEC3G interaction have been described. In this review, we focus on compounds targeting the Integrase-LEDGF/p75 interaction, the Tat-P-TEFb interaction and the Vif-APOBEC3G interaction. Additionally we give an overview of currently discovered compounds presumably targeting cellular cofactor-HIV protein interactions.status: publishe

Lessons learned: HIV points the way towards precision treatment of mixed-lineage leukemia

Protein-protein interactions are involved in most if not all pathogenic and pathophysiological processes and represent attractive therapeutic targets. Extensive biological and clinical research efforts have led to the identification and validation of several cellular hubs that are crucially involved in disease pathogenesis. An interesting example of such a hub is the lens epithelium-derived growth factor (LEDGF/p75), a protein that tethers multiple unrelated proteins and protein complexes to the chromatin. Its chromatin-tethering ability is linked to at least two unrelated diseases-HIV infection and MLL-rearranged acute leukemia. In this review we discuss recent progress in our understanding of the interaction of LEDGF/p75 with its binding partners and focus on the first steps towards therapies targeting protein-protein interactions of LEDGF/p75.publisher: Elsevier articletitle: Lessons Learned: HIV Points the Way Towards Precision Treatment of Mixed-Lineage Leukemia journaltitle: Trends in Pharmacological Sciences articlelink: http://dx.doi.org/10.1016/j.tips.2016.05.005 content_type: article copyright: © 2016 Elsevier Ltd. All rights reserved.status: publishe

Characterization of YB-1 as cellular co-factor of HIV replication

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Fundamental insights into autosomal dominant polycystic kidney disease from human-based cell models

Several animal- and human-derived models are used in autosomal dominant polycystic kidney disease (ADPKD) research to gain insight in the disease mechanism. However, a consistent correlation between animal and human ADPKD models is lacking. Therefore, established human-derived models are relevant to affirm research results and translate findings into a clinical set-up. In this review, we give an extensive overview of the existing human-based cell models. We discuss their source (urine, nephrectomy and stem cell), immortalisation procedures, genetic engineering, kidney segmental origin and characterisation with nephron segment markers. We summarise the most studied pathways and lessons learned from these different ADPKD models. Finally, we issue recommendations for the derivation of human-derived cell lines and for experimental set-ups with these cell lines.status: publishe