scholarly journals rAAV Engineering for Capsid-Protein Enzyme Insertions and Mosaicism Reveals Resilience to Mutational, Structural and Thermal Perturbations

2019 ◽  
Vol 20 (22) ◽  
pp. 5702 ◽  
Author(s):  
Rebecca C. Feiner ◽  
Julian Teschner ◽  
Kathrin E. Teschner ◽  
Marco T. Radukic ◽  
Tobias Baumann ◽  
...  
Keyword(s):  
Biological Function ◽  
Genetic Manipulation ◽  
Full Potential ◽  
Concomitant Increase ◽  
Loop Region ◽  
Viral Particles ◽  
Genetic Level ◽  
Mosaic Approach ◽  
Thermal Perturbations ◽  
Protein Enzyme

Recombinant adeno-associated viruses (rAAV) provide outstanding options for customization and superior capabilities for gene therapy. To access their full potential, facile genetic manipulation is pivotal, including capsid loop modifications. Therefore, we assessed capsid tolerance to modifications of the structural VP proteins in terms of stability and plasticity. Flexible glycine-serine linkers of increasing sizes were, at the genetic level, introduced into the 587 loop region of the VP proteins of serotype 2, the best studied AAV representative. Analyses of biological function and thermal stability with respect to genome release of viral particles revealed structural plasticity. In addition, insertion of the 29 kDa enzyme β-lactamase into the loop region was tested with a complete or a mosaic modification setting. For the mosaic approach, investigation of VP2 trans expression revealed that a Kozak sequence was required to prevent leaky scanning. Surprisingly, even the full capsid modification with β-lactamase allowed for the assembly of capsids with a concomitant increase in size. Enzyme activity assays revealed lactamase functionality for both rAAV variants, which demonstrates the structural robustness of this platform technology.

scholarly journals What is in a Viral Stock: Perspectives and Current Limitations of Flow Virometry

2020 ◽  
Author(s):  
Timothy K. Soh ◽  
Jens B. Bosse
Keyword(s):  
Flow Cytometry ◽  
Full Potential ◽  
Multiple Objects ◽  
Virus Particles ◽  
Viral Particles ◽  
Viral Stock ◽  
Infected Cells ◽  
A Minor ◽  
Unique Capability

Herpesviruses produce a plethora of pleomorphic and heterogeneous particle populations. The composition and biological role of these is not understood. Detailed analysis has been challenging due to the lack of multidimensional identification and purification methodologies. Fluorescence-activated cell sorting (FACS), originally developed to sort objects with at least ten thousand-fold larger volumes, has recently been applied to cellular exosomes as well as viral particles and has been dubbed nanoscale flow cytometry or “flow virometry”. In comparison to other nanoparticles, herpesvirus concentrations can be measured with high precision using simple culturing methods. Here, we used this unique capability to evaluate a standard FACS sorter. We demonstrate that detection and separation capabilities were insufficient to distinguish infectious fluorescent viral populations from populations lacking fluorescence and infectivity. Moreover, fluorescent populations did not contain single virus particles but mostly aggregates. On top, analysis of viral samples by flow cytometry was confounded by swarm detection, as multiple objects are measured simultaneously and interpreted as a single object. Despite these technical difficulties, comparison of crude supernatant to gradient purified HCMV revealed that infectious virus is a minor proportion of the particles released from infected cells. Our data stress the need for a set of standardized controls and protocols when applying FACS to biological nanoparticles and highlights technical challenges that need to be solved before flow virometry can achieve its full potential.

PSIX-29 Study of the association of mtDNA haplogroups and Ages of 100 days in pigs

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 497-497
Author(s):  
Lyubov Getmantseva ◽  
Nekruz Bakoev ◽  
Varvara Shevtsova ◽  
Siroj Bakoev ◽  
Maria Kolosova
Keyword(s):  
Growth Rate ◽  
Production Efficiency ◽  
Mixed Model ◽  
Linear Mixed Model ◽  
Asian Origin ◽  
Mtdna Haplogroups ◽  
Loop Region ◽  
D Loop ◽  
Genetic Level ◽  
Genotype C

Abstract The aim of the work was to determine the mtDNA haplogroups and assess their associations with Days_100 in pigs based on sequencing the D-loop region. The research was carried out on Landrace sows (n = 123). To amplification a fragment of mtDNA D-loop conducted PCR using the following primers: F5 ‘ - TGC AAA CCA AAA CGC CAA GT-3’ and R: 3 ‘ - TTT TTG GGG TTT GGC AAG GC-5. Statistical analyzes were performed using Linear mixed model fit by REML (‘lmerModLmerTest’). The studied group of pigs had the largest number of D haplogroups, which were definitely in 66 sows (53.7%). Haplogroup E was identified in 18 sows (14.6%). Among the haplogroups of Asian origin, which include A, B and C, only genotype C was present. Statistical differences for D100 were established between Hap_C and Hap_E. The presented results indicate the influence of mtDNA haplogroups on Days_100 and pigs of hapogroup E showed the best results compared to analogs of haplogroup E. This may be due to the fact that haplotype E is of European origin, and haplotype C is of Asian origin. Breeding commercial European pigs is focused on increasing the growth rate, and this significantly reduces pig keeping costs and increases production efficiency. It should be noted that changes in growth rate are associated with more intense metabolic processes where mitochondria play a significant role. This may be reflected at the genetic level being determined by the nucleotide sequence of mtDNAs and at the haplotype level in particular. This research was funded by Ministry of Science and Higher Education of the Russian Federation (0445-2021-0008).

scholarly journals Cell geometry across the ring structure of Sitka spruce

2018 ◽  
Vol 15 (142) ◽  
pp. 20180144 ◽  
Author(s):  
T. P. S. Reynolds ◽  
H. C. Burridge ◽  
R. Johnston ◽  
G. Wu ◽  
D. U. Shah ◽  
...  
Keyword(s):  
High Resolution ◽  
Genetic Manipulation ◽  
Cell Structure ◽  
Ring Structure ◽  
Small Scale ◽  
Full Potential ◽  
Micro Computed Tomography ◽  
Cell Geometry ◽  
Structural Size ◽  
Strength And Stiffness

For wood to be used to its full potential as an engineering material, it is necessary to quantify links between its cell geometry and the properties it exhibits at bulk scale. Doing so will make it possible to predict timber properties crucial to engineering, such as mechanical strength and stiffness, and the resistance to fluid flow, and to inform strategies to improve those properties as required, as well as to measure the effects of interventions such as genetic manipulation and chemical modification. Strength, stiffness and permeability of timber all derive from the geometry of its cells, and yet current practice is to predict them based on properties, such as bulk density, that do not directly describe the cell structure. This work explores links between micro-computed tomography data for structural-size pieces of wood, which show the variation of porosity across the wood's ring structure, and high-resolution tomography showing the geometry of the cells, from which we measure cell length, lumen area, porosity, cell wall thickness and the number density of cells. High-resolution scans, while informative, are time-consuming and expensive to run on a large number of samples at the scale of building components. By scanning the same volume of timber at both low and high resolutions (high-resolution scans over a near-continuous volume of timber of approx. 20 mm 3 at 15 μm 3 per voxel), we are able to demonstrate correlations between the measurements at the two different resolutions, reveal the physical basis for these correlations, and demonstrate that the data from the low-resolution scan can be used to estimate the variation in (small-scale) cell geometry throughout a structural-size piece of wood.

Antibiotics: opportunities for genetic manipulation

1989 ◽  
Vol 324 (1224) ◽  
pp. 549-562 ◽  
Keyword(s):  
Genetic Engineering ◽  
Genetic Manipulation ◽  
Antibiotic Production ◽  
Morphological Differentiation ◽  
Basic Structure ◽  
Antibiotic Biosynthesis ◽  
Full Potential ◽  
Metabolite Formation ◽  
Random Mutation

New antibiotics can still be discovered by the development of novel screening procedures. Notable successes over the last few years include the monobactams, β- lactamase inhibitors (clavulanic acid) and new glycopeptides in the antibacterial held; antiparasitic agents such as avermectins; and herbicidal antibiotics like bialaphos. In the future we can expect the engineering of genes from ‘difficult’ pathogens, including mycobacteria and fungi, and cancer cells, to provide increasingly useful in vitro targets for the screening of antibiotics that can kill pathogens and tumours. There will also be a greater awareness of the need to reveal the full potential for antibiotic production on the part of microorganisms by the physiologial and/or genetic awakening of ‘silent’ genes. Nevertheless, the supply of natural antibiotics for direct use or chemical modification is not infinite and there will be increasing scope for widening the range of available antibiotics by genetic engineering. ‘Hybrid’ antibiotics have been shown to be generated by the transfer of genes on suitable vectors between strains producing chemically related compounds. More exciting is the possibility of generating novelty by the genetic engineering of the synthases that determine the basic structure of antibiotics belonging to such classes as the β-lactams and polyketides. Research in this area will certainly yield knowledge of considerable scientific interest and probably also of potential applicability. In the improvement of antibiotic titre in actinomycetes, protoplast fusion between divergent selection lines has taken a place alongside random mutation and screening. In some cases the cloning of genes controlling metabolic ‘bottlenecks’ in fungi and actinomycetes will give an immediate benefit in the conversion of accumulated biosynthetic intermediates to the desired end product. However, the main impact of genetic engineering in titre improvement will probably come only after a further use of this technology to understand and manipulate the regulation of antibiotic biosynthesis as a facet of the general challenge of understanding differential gene expression. Streptomyces offers a particularly fertile field for such research, following the isolation of DNA segments that carry groups of closely linked operons for the biosynthesis of and resistance to particular antibiotics, and of genes with pleiotropic effects on morphological differentiation and secondary metabolite formation.

scholarly journals PostexitSurface Engineering of Retroviral/Lentiviral Vectors

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Christoph Metzner ◽  
Feliks Kochan ◽  
John A. Dangerfield
Keyword(s):  
Chemical Modification ◽  
Genetic Information ◽  
Viral Particle ◽  
Genetic Manipulation ◽  
Regulatory Elements ◽  
Safe Delivery ◽  
Advantages And Disadvantages ◽  
Genomic Level ◽  
Gene Delivery Vectors ◽  
Viral Particles

Gene delivery vectors based on retroviral or lentiviral particles are considered powerful tools for biomedicine and biotechnology applications. Such vectors require modification at the genomic level in the form of rearrangements to allow introduction of desired genes and regulatory elements (genotypic modification) as well as engineering of the physical virus particle (phenotypic modification) in order to mediate efficient and safe delivery of the genetic information to the target cell nucleus. Phenotypic modifications are typically introduced at the genomic level through genetic manipulation of the virus producing cells. However, this paper focuses on methods which allow modification of viral particle surfaces after they have exited the cell, that is, directly on the viral particles in suspension. These methods fall into three categories: (i) direct covalent chemical modification, (ii) membrane-topic reagents, and (iii) adaptor systems. Current applications of such techniques will be introduced and their advantages and disadvantages will be discussed.

scholarly journals Cas9-mediated genome editing in Giardia intestinalis

2021 ◽  
Author(s):  
Vendula Horáčková ◽  
Luboš Voleman ◽  
Markéta Petrů ◽  
Martina Vinopalová ◽  
Filip Weisz ◽  
...  
Keyword(s):  
Genome Editing ◽  
Cell Lines ◽  
Genetic Manipulation ◽  
Protozoan Parasite ◽  
Giardia Intestinalis ◽  
Full Potential ◽  
Knock Out ◽  
Genome Modifications ◽  
First Time

CRISPR/Cas9 system is an extremely powerful technique that is extensively used for different genome modifications in various organisms including parasitic protists. Giardia intestinalis, a protozoan parasite infecting large number of people around the world each year, has been eluding the use of CRISPR/Cas9 technique so far which may be caused by its rather complicated genome containing four copies of each gene in its two nuclei. Apart from only single exception (Ebneter et al., 2016), without the use of CRISPR/Cas9 technology in its full potential, researchers in the field have not been able to establish knock-out cell lines to study the functional aspect of Giardia genes. In this work, we show the ability of in-vitro developed CRISPR/Cas9 components to successfully edit the genome of G. intestinalis. Moreover, we used self-propagating CRISPR/Cas9 system to establish full knock out cell lines for mem, cwp1 and mlf1 genes. We also show that the system function even for essential genes, as we knocked-down tom40, lowering the amount of Tom40 protein by more than 90%. Further, we tested the length of homologous arms needed for successful integration of homology recombination cassette used for genome editing. Taken together, our work introduces CRISPR/Cas9 to Giardia for routine use in the lab, further extending the catalogue of molecular tolls available for genetic manipulation of the protist and allowing researchers to study the function of Giardia genes properly for the first time.

scholarly journals Disrupting HIV-1 capsid formation causes cGAS sensing of viral DNA

2019 ◽  
Author(s):  
Rebecca P. Sumner ◽  
Lauren Harrison ◽  
Emma Touizer ◽  
Thomas P. Peacock ◽  
Matthew Spencer ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Protective Role ◽  
Type I ◽  
Viral Dna ◽  
Innate And Adaptive Immunity ◽  
Capsid Formation ◽  
Diminished Capacity ◽  
Viral Particles ◽  
Hiv 1

SummaryDetection of viral DNA by cyclic GMP-AMP synthase (cGAS) is a first line of defence leading to the production of type-I interferon (IFN). As HIV-1 is not a strong inducer of IFN we have hypothesised that its capsid cloaks viral DNA from cGAS. To test this we generated defective viral particles by treatment with HIV-1 protease inhibitors or by genetic manipulation of gag. These viruses had defective Gag cleavage, reduced infectivity and diminished capacity to saturate TRIM5α. Importantly, unlike wild-type HIV-1, infection with cleavage defective HIV-1 triggered an IFN response in THP-1 cells and primary human macrophages that was dependent on viral DNA and cGAS. Infection in the presence of the capsid destabilising small molecule PF-74 also induced a cGAS-dependent IFN response. These data demonstrate a protective role for capsid and suggest that antiviral activity of capsid- and protease-targeting antivirals may benefit from enhanced innate and adaptive immunity in vivo.

Crystal structures of 7-methylguanosine 5′-triphosphate (m7GTP)- and P1-7-methylguanosine-P3-adenosine-5′,5′-triphosphate (m7GpppA)-bound human full-length eukaryotic initiation factor 4E: biological importance of the C-terminal flexible region

2002 ◽  
Vol 362 (3) ◽  
pp. 539-544 ◽  
Author(s):  
Koji TOMOO ◽  
Xu SHEN ◽  
Koumei OKABE ◽  
Yoshiaki NOZOE ◽  
Shoichi FUKUHARA ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Biological Function ◽  
Full Length ◽  
Initiation Factor ◽  
Regulatory Function ◽  
Structural Basis ◽  
Eukaryotic Initiation Factor ◽  
Eukaryotic Initiation Factor 4E ◽  
Loop Region ◽  
Terminal Loop

The crystal structures of the full-length human eukaryotic initiation factor (eIF) 4E complexed with two mRNA cap analogues [7-methylguanosine 5′-triphosphate (m7GTP) and P1-7-methylguanosine-P3-adenosine-5′,5′-triphosphate (m7GpppA)] were determined at 2.0Å resolution (where 1Å = 0.1nm). The flexibility of the C-terminal loop region of eIF4E complexed with m7GTP was significantly reduced when complexed with m7GpppA, suggesting the importance of the second nucleotide in the mRNA cap structure for the biological function of eIF4E, especially the fixation and orientation of the C-terminal loop region, including the eIF4E phosphorylation residue. The present results provide the structural basis for the biological function of both N- and C-terminal mobile regions of eIF4E in translation initiation, especially the regulatory function through the switch-on/off of eIF4E-binding protein—eIF4E phosphorylation.

Recent Applications of High Resolution Electron Microscopy to Crystalline Arrays of Virus Particles

1978 ◽  
Vol 36 (3) ◽  
pp. 470-482 ◽  
Author(s):  
R.W. Horne
Keyword(s):  
Electron Microscopy ◽  
Image Processing ◽  
Analytical Techniques ◽  
Staining Technique ◽  
High Resolution Electron Microscopy ◽  
Optical Diffraction ◽  
Full Potential ◽  
Virus Particles ◽  
Resolution Electron ◽  
Wide Range

The technique of surrounding virus particles with a neutralised electron dense stain was described at the Fourth International Congress on Electron Microscopy, Berlin 1958 (see Home & Brenner, 1960, p. 625). For many years the negative staining technique in one form or another, has been applied to a wide range of biological materials. However, the full potential of the method has only recently been explored following the development and applications of optical diffraction and computer image analytical techniques to electron micrographs (cf. De Hosier & Klug, 1968; Markham 1968; Crowther et al., 1970; Home & Markham, 1973; Klug & Berger, 1974; Crowther & Klug, 1975). These image processing procedures have allowed a more precise and quantitative approach to be made concerning the interpretation, measurement and reconstruction of repeating features in certain biological systems.

Comparison of Choriocarcinoma and Normal Placenta

1971 ◽  
Vol 29 ◽  
pp. 324-325
Author(s):  
John C. Garancis ◽  
Roland A. Pattillo ◽  
Robert O. Hussa ◽  
Jon V. Straumfjord
Keyword(s):  
Cell Lines ◽  
Small Cells ◽  
Tissue Cultures ◽  
Glycogen Depletion ◽  
Cell Surfaces ◽  
Intercellular Spaces ◽  
Concomitant Increase ◽  
Gestational Choriocarcinoma ◽  
Cytoplasmic Glycogen ◽  
Normal Placenta

Two different cell lines (Be-Wo and Jar) of human gestational choriocarcinoma have been maintained in continuous tissue culture for a period of four and two years respectively without losing the ability to elaborate human chorionic gonadotropin (HCG). Tissue cultures, as revealed by electron microscopy, consisted of small cells with single nuclei. In some instances cell surfaces were provided with microvilli but more often the intercellular spaces were narrow and bridged by desmosomes. However, syncytium was not formed. Endoplasmic reticulum (ER) was poorly developed in both cell lines, except in some Be-Wo cells it was prominent. Golgi complex, lysosomes and numerous free ribosomes, as well as excessive cytoplasmic glycogen, were present in all cells (Fig. 1). Glycogen depletion and concomitant increase of ER were observed in many cells following a single dose of 10 ugm/ml of adrenalin added to medium (Fig. 2).

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