22 research outputs found
The Rev/Rex homolog HERV-K cORF multimerizes via a C-terminal domain
AbstractExpression of human endogenous retrovirus K (HERV-K) is associated with germ-cell neoplasia. HERV-K encodes a protein of the Rev/Rex family, cORF, that supports cellular transformation and binds the promyelocytic leukemia zinc finger (PLZF) protein implicated in spermatogenesis. Rev/Rex function invariably depends on multimerization. Here we show that cORF likewise self-associates to form higher-order oligomers. Amino acids (aa) 47â87 in cORF are sufficient, aa 75â87 essential for self-association. Consistently, this domain is predicted to form a hydrophobic α-helix that may represent an oligomerization interface. The existence of a dimerization-competent cORF mutant lacking PLZF-binding activity (cORF47â87) suggests a way of dominant negative inhibition of the proposed tumor susceptibility factor cORF
Co-Delivery of mRNA and pDNA Using Thermally Stabilized Coacervate-Based Core-Shell Nanosystems
Co-delivery of different species of protein-encoding polynucleotides, e.g., messenger RNA
(mRNA) and plasmid DNA (pDNA), using the same nanocarrier is an interesting topic that remains
scarcely researched in the field of nucleic acid delivery. The current study hence aims to explore the
possibility of the simultaneous delivery of mRNA (mCherry) and pDNA (pAmCyan) using a single
nanocarrier. The latter is based on gelatin type A, a biocompatible, and biodegradable biopolymer of
broad pharmaceutical application. A core-shell nanostructure is designed with a thermally stabilized
gelatinâpDNA coacervate in its center. Thermal stabilization enhances the coreâs colloidal stability
and pDNA shielding effect against nucleases as confirmed by nanoparticle tracking analysis and gel
electrophoresis, respectively. The stabilized, pDNA-loaded core is coated with the cationic peptide
protamine sulfate to enable additional surface-loading with mRNA. The dual-loaded core-shell
system transfects murine dendritic cell line DC2.4 with both fluorescent reporter mRNA and pDNA
simultaneously, showing a transfection efficiency of 61.4 ± 21.6% for mRNA and 37.6 ± 19.45% for
pDNA, 48 h post-treatment, whereas established commercial, experimental, and clinical transfection
reagents fail. Hence, the unique co-transfectional capacity and the negligible cytotoxicity of the
reported system may hold prospects for vaccination among other downstream applications
Ini1/hSNF5 is dispensable for retrovirus-induced cytoplasmic accumulation of PML and does not interfere with integration.
International audienceRetroviral infection triggers the cytoplasmic translocation of two Crm1-dependent shuttle factors, namely the Ini1 (integrase interactor 1, hSNF5) and the promyelocytic leukemia (PML) protein. Blocking nuclear export of shuttle factors by leptomycin B increases the efficiency of retroviral integration, suggesting that some may mediate antiviral activity. While PML was shown to counteract proviral establishment, it remained unclear whether Ini1, a protein implicated in various processes during human immunodeficiency virus replication, has the same potential. Employing RNA interference-mediated knock-down of Ini1, we show here that the simultaneous accumulation of both proteins in the cytoplasm likely reflects two non-interdependent phenomena. Furthermore, Ini1 does not interfere with retroviral integration, as cells lacking Ini1 show no increased infection susceptibility
Co-Delivery of mRNA and pDNA Using Thermally Stabilized Coacervate-Based Core-Shell Nanosystems.
Co-delivery of different species of protein-encoding polynucleotides, e.g., messenger RNA (mRNA) and plasmid DNA (pDNA), using the same nanocarrier is an interesting topic that remains scarcely researched in the field of nucleic acid delivery. The current study hence aims to explore the possibility of the simultaneous delivery of mRNA (mCherry) and pDNA (pAmCyan) using a single nanocarrier. The latter is based on gelatin type A, a biocompatible, and biodegradable biopolymer of broad pharmaceutical application. A core-shell nanostructure is designed with a thermally stabilized gelatin-pDNA coacervate in its center. Thermal stabilization enhances the core's colloidal stability and pDNA shielding effect against nucleases as confirmed by nanoparticle tracking analysis and gel electrophoresis, respectively. The stabilized, pDNA-loaded core is coated with the cationic peptide protamine sulfate to enable additional surface-loading with mRNA. The dual-loaded core-shell system transfects murine dendritic cell line DC2.4 with both fluorescent reporter mRNA and pDNA simultaneously, showing a transfection efficiency of 61.4 ± 21.6% for mRNA and 37.6 ± 19.45% for pDNA, 48 h post-treatment, whereas established commercial, experimental, and clinical transfection reagents fail. Hence, the unique co-transfectional capacity and the negligible cytotoxicity of the reported system may hold prospects for vaccination among other downstream applications
Co-Delivery of mRNA and pDNA Using Thermally Stabilized Coacervate-Based Core-Shell Nanosystems
Co-delivery of different species of protein-encoding polynucleotides, e.g., messenger RNA (mRNA) and plasmid DNA (pDNA), using the same nanocarrier is an interesting topic that remains scarcely researched in the field of nucleic acid delivery. The current study hence aims to explore the possibility of the simultaneous delivery of mRNA (mCherry) and pDNA (pAmCyan) using a single nanocarrier. The latter is based on gelatin type A, a biocompatible, and biodegradable biopolymer of broad pharmaceutical application. A core-shell nanostructure is designed with a thermally stabilized gelatinâpDNA coacervate in its center. Thermal stabilization enhances the coreâs colloidal stability and pDNA shielding effect against nucleases as confirmed by nanoparticle tracking analysis and gel electrophoresis, respectively. The stabilized, pDNA-loaded core is coated with the cationic peptide protamine sulfate to enable additional surface-loading with mRNA. The dual-loaded core-shell system transfects murine dendritic cell line DC2.4 with both fluorescent reporter mRNA and pDNA simultaneously, showing a transfection efficiency of 61.4 ± 21.6% for mRNA and 37.6 ± 19.45% for pDNA, 48 h post-treatment, whereas established commercial, experimental, and clinical transfection reagents fail. Hence, the unique co-transfectional capacity and the negligible cytotoxicity of the reported system may hold prospects for vaccination among other downstream applications
A novel specific edge effect correction method for RNA interference screenings.
International audienceMOTIVATION: High-throughput screening (HTS) is an important method in drug discovery in which the activities of a large number of candidate chemicals or genetic materials are rapidly evaluated. Data are usually obtained by measurements on samples in microwell plates and are often subjected to artefacts that can bias the result selection. We report here a novel edge effect correction algorithm suitable for RNA interference (RNAi) screening, because its normalization does not rely on the entire dataset and takes into account the specificities of such a screening process. The proposed method is able to estimate the edge effects for each assay plate individually using the data from a single control column based on diffusion model, and thus targeting a specific but recurrent well-known HTS artefact. This method was first developed and validated using control plates and was then applied to the correction of experimental data generated during a genome-wide siRNA screen aimed at studying HIV-host interactions. The proposed algorithm was able to correct the edge effect biasing the control data and thus improve assay quality and, consequently, the hit-selection step
A pulmonary mucus surrogate for investigating antibiotic permeation and activity against Pseudomonas aeruginosa biofilms.
A growing number of studies show that innate immune cells can undergo functional reprogramming, facilitating a faster and enhanced response to heterologous secondary stimuli. This concept has been termed "trained immunity." We outline here a protocol to recapitulate this in vitro using adherent monocytes from consecutive isolation of peripheral blood mononuclear cells. The induction of trained immunity and the associated functional reprogramming of monocytes is described in detail using ÎČ-glucan (from Candida albicans) and Bacillus Calmette-GuĂ©rin as examples. For complete details on the use and execution of this protocol, please refer to Repnik et al. (2003) and Bekkering et al. (2016)
Transferring Microclusters of P. aeruginosa Biofilms to the Air : Liquid Interface of Bronchial Epithelial Cells for Repeated Deposition of Aerosolized Tobramycin
As an alternative to technically demanding and ethically
debatable animal models, the use of organotypic and disease-relevant
human cell culture models may improve the throughput, speed, and
success rate for the translation of novel anti-infectives into the clinic.
Besides bacterial killing, host cell viability and barrier function appear as
relevant but seldomly measured readouts. Moreover, bacterial virulence
factors and signaling molecules are typically not addressed in current cell
culture models. Here, we describe a reproducible protocol for cultivating
barrier-forming human bronchial epithelial cell monolayers on Transwell
inserts and infecting them with microclusters of pre-grown mature
Pseudomonas aeruginosa PAO1 biofilms under the airâliquid interface
conditions. Bacterial growth and quorum sensing molecules were
determined upon tobramycin treatment. The host cell response was
simultaneously assessed through cell viability, epithelial barrier function, and cytokine release. By repeated deposition of aerosolized
tobramycin after 1, 24, and 48 h, bacterial growth was controlled (reduction from 10 to 4 log10 CFU/mL), which leads to epithelial
cell survival for up to 72 h. E-cadherinâs cellâcell adhesion protein expression was preserved with the consecutive treatment, and
quorum sensing molecules were reduced. However, the bacteria could not be eradicated and epithelial barrier function was impaired,
similar to the currently observed situation in the clinic in lack of more efficient anti-infective therapies. Such a human-based in vitro
approach has the potential for the preclinical development of novel anti-infectives and nanoscale delivery systems for oral inhalation
Automated genome-wide visual profiling of cellular proteins involved in HIV infection.
International audienceRecent genome-wide RNAi screens have identified >842 human genes that affect the human immunodeficiency virus (HIV) cycle. The list of genes implicated in infection differs between screens, and there is minimal overlap. A reason for this variance is the interdependence of HIV infection and host cell function, producing a multitude of indirect or pleiotropic cellular effects affecting the viral infection during RNAi screening. To overcome this, the authors devised a 15-dimensional phenotypic profile to define the viral infection block induced by CD4 silencing in HeLa cells. They demonstrate that this phenotypic profile excludes nonspecific, RNAi-based side effects and viral replication defects mediated by silencing of housekeeping genes. To achieve statistical robustness, the authors used automatically annotated RNAi arrays for seven independent genome-wide RNAi screens. This identified 56 host genes, which reliably reproduced CD4-like phenotypes upon HIV infection. The factors include 11 known HIV interactors and 45 factors previously not associated with HIV infection. As proof of concept, the authors confirmed that silencing of PAK1, Ku70, and RNAseH2A impaired HIV replication in Jurkat cells. In summary, multidimensional, visual profiling can identify genes required for HIV infection