scholarly journals Development and Characterization of Novel Empty Adenovirus Capsids and Their Impact on Cellular Gene Expression

2003 ◽  
Vol 77 (23) ◽  
pp. 12881-12885 ◽  
Author(s):  
Jackie L. Stilwell ◽  
Douglas M. McCarty ◽  
Atsuko Negishi ◽  
Richard Superfine ◽  
R. Jude Samulski
Keyword(s):  
Viral Entry ◽  
First Generation ◽  
Viral Vector ◽  
Intact Virion ◽  
Force Microscopy ◽  
High Particle ◽  
Atomic Force ◽  
Host Innate Immune Response ◽  
Confocal Images ◽  
Infected Cells

ABSTRACT Adenovirus (Ad) has been extensively studied as a eukaryotic viral vector. As these vectors have evolved from first-generation vectors to vectors that contain either very few or no viral genes (“gutless” Ad), significant reductions in the host innate immune response upon infection have been observed. Regardless of these vector improvements an unknown amount of toxicity has been associated with the virion structural proteins. Here we demonstrate the ability to generate high particle numbers (1011 to 1012) of Ad empty virions based on a modification of Cre/lox gutless Ad vectors. Using a battery of analyses (electron microscopy, atomic force microscopy, confocal images, and competition assays) we characterized this reagent and determined that it (i) makes intact virion particles, (ii) competes for receptor binding with wild-type Ad, and (iii) enters the cell proficiently, demonstrating an ability to carry out essential steps of viral entry. To further study the biological impact of these Ad empty virions on infected cells, we carried out DNA microarray analysis. Compared to that for recombinant Ad, the number of mRNAs modulated upon infection was significantly reduced but the expression signatures were similar. This reagent provides a valuable tool for studies of Ad in that researchers can examine the effect of infection in the presence of the virion capsid alone.

scholarly journals Bursting out: linking changes in nanotopography and biomechanical properties of biofilm-forming Escherichia coli to the T4 lytic cycle

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Shiju Abraham ◽  
Yair Kaufman ◽  
François Perreault ◽  
Ry Young ◽  
Edo Bar-Zeev
Keyword(s):  
Cell Envelope ◽  
Biomechanical Properties ◽  
Lytic Cycle ◽  
Planktonic Cells ◽  
Force Microscopy ◽  
Bacteriophage Infection ◽  
Atomic Force ◽  
Biofilm Removal ◽  
Infected Cells ◽  
The Impact

AbstractThe bacteriophage infection cycle has been extensively studied, yet little is known about the nanostructure and mechanical changes that lead to bacterial lysis. Here, atomic force microscopy was used to study in real time and in situ the impact of the canonical phage T4 on the nanotopography and biomechanics of irreversibly attached, biofilm-forming E. coli cells. The results show that in contrast to the lytic cycle in planktonic cells, which ends explosively, anchored cells that are in the process of forming a biofilm undergo a more gradual lysis, developing distinct nanoscale lesions (~300 nm in diameter) within the cell envelope. Furthermore, it is shown that the envelope rigidity and cell elasticity decrease (>50% and >40%, respectively) following T4 infection, a process likely linked to changes in the nanostructure of infected cells. These insights show that the well-established lytic pathway of planktonic cells may be significantly different from that of biofilm-forming cells. Elucidating the lysis paradigm of these cells may advance biofilm removal and phage therapeutics.

scholarly journals A Computational Framework to Model Mesenchymal Stem Cell Nucleus Mechanics Using Confocal Microscopy

2020 ◽  
Author(s):  
Zeke Kennedy
Keyword(s):  
Atomic Force Microscopy ◽  
Mechanical Testing ◽  
Cell Nucleus ◽  
Mechanical Stimuli ◽  
Computational Framework ◽  
Conversion Factors ◽  
Testing Apparatus ◽  
Force Microscopy ◽  
Atomic Force ◽  
Confocal Images

The mechanical properties of the cell nucleus are emerging as a key component in genetic transcription. It has been shown that the stiffness of the nucleus in part regulates the transcription of genes in response to external mechanical stimuli. The stiffness has been shown to change as a result of both disease and changes to the external environment. While the mechanical structure of the nucleus can be visually documented using a confocal microscope, it is currently impossible to test the stiffness of the nucleus without a mechanical testing apparatus such as an atomic force microscope. This is problematic in that the use of a mechanical testing apparatus involves deconstructing the cell in order to isolate the nucleus and is unable to provide data on internal heterochromatin dynamics within the nucleus. Therefore, our research focused on developing a computational framework that would allow researchers to model the mechanical contributions of the nucleus specific geometry and material dispersion of both chromatin and LaminA/C within an individual nucleus in order to improve the ability of researchers to study the nucleus. We began by developing a procedure that could generate a finite element geometry of a nucleus using confocal images. This procedure was then utilized to generate models that contained elasticity values that corresponded to the voxel intensities of images of both chromatin and LaminA/C by using a set of conversion factors to link image voxel intensity to model stiffness. We then tuned these conversion factors by running in silico atomic force microscopy experiments on these models while comparing the simulation results to atomic force microscopy data from real world nuclei. From this experiment we were able to find a set of conversion factors that allowed us to replicate the external response of the nucleus. Our developed computational framework will allow future researchers to study the contribution of multitude of sub-nuclear structures and predict global nuclear stiffness of multiple nuclei based on confocal images and AFM tests.

scholarly journals Lipid specificity of the immune effector perforin

2020 ◽  
Author(s):  
Adrian W. Hodel ◽  
Jesse A. Rudd-Schmidt ◽  
Joseph A. Trapani ◽  
Ilia Voskoboinik ◽  
Bart W. Hoogenboom
Keyword(s):  
Pore Formation ◽  
Immune Effector ◽  
Force Microscopy ◽  
Atomic Force ◽  
Residual Protein ◽  
Dense Membrane ◽  
Lipid Species ◽  
Infected Cells ◽  
Lipid Specificity ◽  
Insight Into

AbstractPerforin is a pore forming protein used by cytotoxic T lymphocytes to remove cancerous or virus-infected cells during immune response. During the response, the lymphocyte membrane becomes refractory to perforin function by accumulating densely ordered lipid rafts and externalizing negatively charged lipid species. The dense membrane packing lowers the capacity of perforin to bind, and negatively charged lipids scavenge any residual protein before pore formation. Using atomic force microscopy on model membrane systems, we here provide insight into the molecular basis of perforin lipid specificity.

scholarly journals The engineered peptide construct NCAM1-Aβ inhibits aggregation of the human prion protein (PrP)

2021 ◽  
Author(s):  
Maciej Gielnik ◽  
Lilia Zhukova ◽  
Igor Zhukov ◽  
Astrid Gräslund ◽  
Maciej Kozak ◽  
...  
Keyword(s):  
Prion Protein ◽  
Molecular Mechanisms ◽  
Prion Diseases ◽  
Disease Treatment ◽  
Force Microscopy ◽  
Atomic Force ◽  
Afm Imaging ◽  
Human Prion Protein ◽  
Infected Cells ◽  
Treatment Procedures

AbstractIn prion diseases, the prion protein (PrP) becomes misfolded and forms fibrillar aggregates, which are resistant to proteinase degradation and become responsible for prion infectivity and pathology. So far, no drug or treatment procedures have been approved for prion disease treatment. We have previously shown that engineered cell-penetrating peptide constructs can reduce the amount of prion aggregates in infected cells. The molecular mechanisms underlying this effect are however unknown. Here, we use atomic force microscopy (AFM) imaging to show that the aggregation of the human PrP protein can be inhibited by equimolar amounts of the 25 residues long engineered peptide construct NCAM1-Aβ.

Sign in / Sign up

Export Citation Format

Share Document