scholarly journals 210. Modelling Viral Insertion Sites Via Network Based Frameworks

2013 ◽  
Vol 21 ◽  
pp. S81
Keyword(s):  
Insertion Sites ◽  
Viral Insertion

scholarly journals PCIP-seq: simultaneous sequencing of integrated viral genomes and their insertion sites with long reads

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Maria Artesi ◽  
Vincent Hahaut ◽  
Basiel Cole ◽  
Laurens Lambrechts ◽  
Fereshteh Ashrafi ◽  
...  
Keyword(s):  
Viral Genome ◽  
Clonal Expansion ◽  
Endogenous Retroviruses ◽  
Viral Genomes ◽  
Short Read Sequencing ◽  
Long Reads ◽  
Infected Cells ◽  
Insertion Sites ◽  
Viral Insertion ◽  
Hiv 1

AbstractThe integration of a viral genome into the host genome has a major impact on the trajectory of the infected cell. Integration location and variation within the associated viral genome can influence both clonal expansion and persistence of infected cells. Methods based on short-read sequencing can identify viral insertion sites, but the sequence of the viral genomes within remains unobserved. We develop PCIP-seq, a method that leverages long reads to identify insertion sites and sequence their associated viral genome. We apply the technique to exogenous retroviruses HTLV-1, BLV, and HIV-1, endogenous retroviruses, and human papillomavirus.

Abstract 257: Bone Marrow Cells Form New Cardiomyocytes in the Infarcted Myocardium

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Fumihiro Sanada ◽  
Junghyung Kim ◽  
Anna Czarna ◽  
Sergio Signore ◽  
Andrea Sorrentino ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Fluorescent Proteins ◽  
Infarcted Myocardium ◽  
Multiple Cell ◽  
Insertion Sites ◽  
Color Combinations ◽  
Viral Insertion ◽  
Marrow Cells

The ability of c-kit-positive bone marrow cells (c-kit-BMCs) to form cardiomyocytes following delivery to the acutely infarcted myocardium is a matter of debate. In an attempt to resolve the controversy, we raised the possibility that c-kit-BMCs represent a functionally heterogeneous pool, containing cells with distinct transdifferentiation potential. To test this hypothesis, c-kit-BMCs were infected with EGFP-lentiviruses and injected in infarcted hearts. At 1 and 2 weeks, the regenerated myocardium was enzymatically digested and EGFP-labeled myocytes, endothelial cells (ECs), fibroblasts, and c-kit-cells were sorted. By employing a PCR-based method of detection of viral integrants, we searched for unique sites of viral insertion in the infected c-kit-BMCs and their progeny. Common insertion sites were found in the DNA of all cell populations, documenting that single c-kit-BMCs transdifferentiated into multiple cell lineages. However, the 31 detected clones shared 9 distinct sites of integration, suggesting that myocytes in different animals were generated by specific subsets of c-kit-BMCs, which retained the ability to transdifferentiate. To strengthen the observation that myocardial regeneration is oligoclonal in nature, freshly isolated FACS-sorted c-kit-BMCs were simultaneously transduced with 3 lentiviruses, each encoding red, green or blue (RGB) fluorescent proteins. Different combinations of inserted vectors resulted in the formation of numerous c-kit-BMC clones of mixed colors. RGB-infected HSCs were injected in infarcted rats; at 4 days, engrafted c-kit-BMCs showed a polyclonal pattern characterized by the presence of cells labeled by most of the color combinations seen in vitro. However, at 2 weeks, only a few of the homogeneously colored colonies seen at 4 days persisted and formed large patches of cells composed of myocytes, ECs, and smooth muscle cells. Our findings document that c-kit-BMCs are functionally heterogeneous and have different ability to survive, engraft, and transdifferentiate in the ischemic heart. At present, clonal c-kit-BMCs are being analyzed by RNA-sequencing for the identification of the unique phenotypical properties of c-kit-BMCs with high transdifferentiation potential.

scholarly journals HGT-ID: an efficient and sensitive workflow to detect human-viral insertion sites using next-generation sequencing data

2018 ◽  
Vol 19 (1) ◽  
Author(s):  
Saurabh Baheti ◽  
Xiaojia Tang ◽  
Daniel R. O’Brien ◽  
Nicholas Chia ◽  
Lewis R. Roberts ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Next Generation Sequencing Data ◽  
Next Generation ◽  
Sequencing Data ◽  
Insertion Sites ◽  
Viral Insertion ◽  
Generation Sequencing

scholarly journals VISMapper: ultra-fast exhaustive cartography of viral insertion sites for gene therapy

2017 ◽  
Vol 18 (1) ◽  
Author(s):  
José M. Juanes ◽  
Asunción Gallego ◽  
Joaquín Tárraga ◽  
Felipe J. Chaves ◽  
Pablo Marín-Garcia ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Insertion Sites ◽  
Viral Insertion

scholarly journals Viral Insertion Safety in Patients with Glioblastoma Who Received a Novel Lentiviral MGMT-P140K Gene Therapy to Protect Bone Marrow from Chemotherapy: No Dominant Clonal Evolution Observed with Chemo-Selection

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4801-4801
Author(s):  
Hua Fung ◽  
Andrew E Sloan ◽  
Jane Reese ◽  
Basem M. William ◽  
Karen Lingas ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Lentiviral Vector ◽  
Lymphoblastic Leukemia ◽  
Insertion Site ◽  
Retroviral Vectors ◽  
Insertion Mutagenesis ◽  
Drug Selection ◽  
Insertion Sites ◽  
Viral Insertion

Abstract INTRODUCTION: To protect normal bone marrow from chemotherapy in glioblastoma patients, we have developed a novel strategy by introducing a strong DNA repair protein, mutant (P140K) of human methylguanine methyltransferase (MGMT), into patients’ CD34+ hematopoietic progenitors (HPC) by lentiviral gene transfer leading to selective expansion of drug-resistant P140K-MGMT CD34+ cells and their myeloid and immune cell progeny. METHODS: To achieve long-term stable expression of the P140K-MGMT gene, we used a lentiviral vector which integrates into the host genome. However, viral insertion mutagenesis has raised safety concerns; as the previous γ-retroviral vectors were associated with insertion mutations leading to development of acute lymphoblastic leukemia in 20% of treated patients. Nevertheless, new improved lentiviral vector with safe feature of insertion site far away from gene transcription start site has been developed. Here we evaluated the safety of a lentivirus vector under selection pressure of chemotherapy. HYPOTHESIS: Our lentiviral vector is safer than traditional γ-retroviral vectors as evident by lack of early clonal dominance even with a chemo-selection. RESULTS: Three glioblastoma patients were recruited and underwent resection, after which CD34+ HPC were mobilized with filgrastim, isolated by magnetic bead separation (Miltneyi CliniMACS), and transduced ex vivo with a GMP-grade lentiviral P140K-MGMT vector (Lentigen Corp). Subsequently, patients received radiation/temozolomide for 6 weeks and up to five cycles of monthly O6-benzylguanine/temozolomide (BG/TMZ) treatment. As a result, all three patients demonstrated a 5-15 fold selective expansion of P140K-MGMT positive HPC and their progeny granulocyte and mononuclear cells in peripheral blood and a small number of CFUs from bone marrow indicating a drug-selection mechanism. The viral insertion sites in the cells of these three patients were closely monitored in each chemotherapy cycle and the patients were followed for up to 1 year after the last therapy. We mapped a total of 17,308 viral insertion sites (VIS), for patient 1(6,146), patient 2(2,081) and patient 3(9,081) and the unique viral insertion sites (UVIS) was 382, i.e. 135, 76 and 171 for patient 1, patient 2 and patient 3 respectively. Overall, during the drug-treatment period, there were no persistent UVIS. Moreover, at the conclusion of BG/TMZ treatment, the VIS number sharply diminished. CONCLUSION: Gene transfer of LV MGMTP140K vector into hematopoietic progenitor cells did not lead to clonal dominance during or after drug selection. Dose escalation of BG/TMZ will define hematopoietic tolerance and treatment response. Disclosures Embree: Lentigen Technology Inc: Employment. Dropulic:Lentigen Technology Inc: Employment, Patents & Royalties.

scholarly journals Automatic modelling of human musculoskeletal ligaments – Framework overview and model quality evaluation

2021 ◽  
pp. 1-14
Author(s):  
Noura Hamze ◽  
Lukas Nocker ◽  
Nikolaus Rauch ◽  
Markus Walzthöni ◽  
Matthias Harders ◽  
...  
Keyword(s):  
Statistical Model ◽  
Soft Tissues ◽  
Automatic Generation ◽  
Cadaveric Study ◽  
Patient Specific ◽  
Mean Square ◽  
Shape Modelling ◽  
Morphological Studies ◽  
Insertion Sites ◽  
Mean Square Errors

BACKGROUND: Accurate segmentation of connective soft tissues in medical images is very challenging, hampering the generation of geometric models for bio-mechanical computations. Alternatively, one could predict ligament insertion sites and then approximate the shapes, based on anatomical knowledge and morphological studies. OBJECTIVE: In this work, we describe an integrated framework for automatic modelling of human musculoskeletal ligaments. METHOD: We combine statistical shape modelling with geometric algorithms to automatically identify insertion sites, based on which geometric surface/volume meshes are created. As clinical use case, the framework has been applied to generate models of the forearm interosseous membrane. Ligament insertion sites in the statistical model were defined according to anatomical predictions following a published approach. RESULTS: For evaluation we compared the generated sites, as well as the ligament shapes, to data obtained from a cadaveric study, involving five forearms with 15 ligaments. Our framework permitted the creation of models approximating ligaments’ shapes with good fidelity. However, we found that the statistical model trained with the state-of-the-art prediction of the insertion sites was not always reliable. Average mean square errors as well as Hausdorff distances of the meshes could increase by an order of magnitude, as compared to employing known insertion locations of the cadaveric study. Using those, an average mean square error of 0.59 mm and an average Hausdorff distance of less than 7 mm resulted, for all ligaments. CONCLUSIONS: The presented approach for automatic generation of ligament shapes from insertion points appears to be feasible but the detection of the insertion sites with a SSM is too inaccurate, thus making a patient-specific approach necessary.

scholarly journals The use of retrotransposons as markers for mapping genes responsible for fitness differences between related Drosophila melanogaster strains

1993 ◽  
Vol 62 (2) ◽  
pp. 125-131 ◽  
Author(s):  
Sergey V. Nuzhdin ◽  
Peter D. Keightley ◽  
Elena G. Pasyukova
Keyword(s):  
Drosophila Melanogaster ◽  
Molecular Markers ◽  
Maximum Likelihood ◽  
Quantitative Trait ◽  
Pericentromeric Region ◽  
Related Strain ◽  
Gene Frequencies ◽  
Ha Gene ◽  
Insertion Sites ◽  
Additional Locus

SummaryHitch-hiking of dispersed mobile elements serving as molecular markers was used as a new tool for mapping quantitative trait loci in Drosophila melanogaster. Two Drosophila strains with high fitness (HA) were backcrossed repeatedly to a closely related strain with low fitness (LA) to initiate experimental populations with expected HA gene frequencies of 1/32. The frequencies of 19 insertion sites of the retrotransposons mdg1 and copia were analyzed after 11 to 17 generations. Frequencies of sites from the HA line increased substantially in the pericentromeric region, indicating that one or more loci responsible for the fitness difference between the strains were located there. A maximum likelihood (ML) procedure was applied to estimate selection coefficients associated with the markers, and this indicated a broad, strongly selected region of the chromosome. At least one additional locus was localized in the middle of the 2L arm. Possible applications of this method are discussed.

scholarly journals Tn5406, a New Staphylococcal Transposon Conferring Resistance to Streptogramin A and Related Compounds Including Dalfopristin

2002 ◽  
Vol 46 (8) ◽  
pp. 2337-2343 ◽  
Author(s):  
Julien Haroche ◽  
Jeanine Allignet ◽  
Névine El Solh
Keyword(s):  
Staphylococcus Aureus ◽  
Insertion Site ◽  
Single Copy ◽  
Amino Acid Identity ◽  
Plasmid Copy ◽  
Acid Identity ◽  
Abc Protein ◽  
Single Plasmid ◽  
Insertion Sites ◽  
Related Compounds

ABSTRACT We characterized a new transposon, Tn5406 (5,467 bp), in a clinical isolate of Staphylococcus aureus (BM3327). It carries a variant of vgaA, which encodes a putative ABC protein conferring resistance to streptogramin A but not to mixtures of streptogramins A and B. It also carries three putative genes, the products of which exhibit significant similarities (61 to 73% amino acid identity) to the three transposases of the staphylococcal transposon Tn554. Like Tn554, Tn5406 failed to generate target repeats. In BM3327, the single copy of Tn5406 was inserted into the chromosomal att554 site, which is the preferential insertion site of Tn554. In three other independent S. aureus clinical isolates, Tn5406 was either present as a single plasmid copy (BM3318), as two chromosomal copies (BM3252), or both in the chromosome and on a plasmid (BM3385). The Tn5406-carrying plasmids also contain two other genes, vgaB and vatB. The insertion sites of Tn5406 in BM3252 were studied: one copy was in att554, and one copy was in the additional SCCmec element. Amplification experiments revealed circular forms of Tn5406, indicating that this transposon might be active. To our knowledge, a transposon conferring resistance to streptogramin A and related compounds has not been previously described.

Integration of viral DNA sequences in cells transformed by adenovirus 2 or SV40

1980 ◽  
Vol 210 (1180) ◽  
pp. 423-435 ◽  
Keyword(s):  
Cell Lines ◽  
Dna Sequences ◽  
Viral Genome ◽  
Viral Dna ◽  
Viral Genomes ◽  
Viral Dnas ◽  
Cellular Dna ◽  
Viral Insertion ◽  
Viral Sequences ◽  
Heteroduplex Formation

We have cloned and propagated in prokaryotic vectors the viral DNA sequences that are integrated in a variety of cells transformed by adenovirus 2 or SV40. Analysis of the clones reveals that the viral DNA sequences sometimes are arranged in a simple fashion, collinear with the viral genome; in other cell lines there are complex arrangements of viral sequences in which tracts of the viral genome are inverted with respect to each other. In several cases the nucleotide sequences at the joints between cell and viral sequences have been determined: usually there is a sharp transition between cellular and viral DNAs. The viral sequences are integrated at different locations within the genomes of different cell lines; likewise there is no specific site on the viral genomes at which integration occurs. Sometimes the viral sequences are integrated within repetitive cellular DNA, and sometimes within unique sequences. In some cases there is evidence that the viral sequences along with the flanking cell DNA have been amplified after integration. The sequences that flank the viral insertion in the line of SV40-transformed rat cells known as 14B have been used as probes to isolate, from untransformed rat cells, clones that carry the region of the chromosome in which integration occurred. Analysis of the structure of these clones by restriction endonuclease digestion and heteroduplex formation shows that a rearrangement of cellular sequences has occurred, presumably as a consequence of integration.

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