Journal: Nucleus

Article Title: A novel cell permeable DNA replication and repair marker

doi: 10.4161/nucl.36290

Figure Lengend Snippet: Design of PCNA interacting peptides (PIP). The red box highlights the residues binding to the PCNA binding pocket. The dotted lines indicate the consensus mutations introduced on the p21 141–157 peptide to optimize the PIP-box sequence and obtain the p21–08 peptide. The IFS (p21 158–160 ) sequence from p21 was removed to reduce the binding between the p21–08 and the CDK-cyclin and make its binding more selective toward PCNA. Additionally a previously described synthetic peptide PL derived from Pogo and DNA ligase I was used.

Article Snippet: The sequences of the p21 peptide and the two highest PCNA binding affinity peptides p21–08 and PL screened are displayed. ( B ) Structures of the p21 peptides and the two PIPs, i.e., p21–08 and PL sequences obtained from molecular dynamics simulations are shown. ( C ) Images showing that these peptides ([F-p21], [F-p21–08], [F-PL]) after microinjection clearly co-localize with GFP-PCNA at replication sites. ( D ) When the peptides are added to the extracellular media they are not able to enter the cells.

Techniques: Binding Assay, Sequencing, Derivative Assay

Journal: Nucleus

Article Title: A novel cell permeable DNA replication and repair marker

doi: 10.4161/nucl.36290

Figure Lengend Snippet: PCNA interacting peptides bind to PCNA in vivo but are not cell permeable. ( A ) Crystal structure of the PCNA trimer (green) with the C-terminus region of the p21 protein (red) bound to it (PDB ID 1AXC). The sequences of the p21 peptide and the two highest PCNA binding affinity peptides p21–08 and PL screened are displayed. ( B ) Structures of the p21 peptides and the two PIPs, i.e., p21–08 and PL sequences obtained from molecular dynamics simulations are shown. ( C ) Images showing that these peptides ([F-p21], [F-p21–08], [F-PL]) after microinjection clearly co-localize with GFP-PCNA at replication sites. ( D ) When the peptides are added to the extracellular media they are not able to enter the cells. The abbreviation PC stands for phase contrast and DIC for differential interference contrast. Scale bar 5 μm.

Article Snippet: The sequences of the p21 peptide and the two highest PCNA binding affinity peptides p21–08 and PL screened are displayed. ( B ) Structures of the p21 peptides and the two PIPs, i.e., p21–08 and PL sequences obtained from molecular dynamics simulations are shown. ( C ) Images showing that these peptides ([F-p21], [F-p21–08], [F-PL]) after microinjection clearly co-localize with GFP-PCNA at replication sites. ( D ) When the peptides are added to the extracellular media they are not able to enter the cells.

Techniques: In Vivo, Binding Assay, Microinjection

Journal: Nucleus

Article Title: A novel cell permeable DNA replication and repair marker

doi: 10.4161/nucl.36290

Figure Lengend Snippet: Rationale to develop a cell permeable DNA replication and repair marker by targeting PCNA. ( A ) The nucleus of a cell expressing fluorescently labeled PCNA was microirradiated. PCNA is recruited to damaged DNA sites. Fusion of the PIP with a CPP [F -TAT-p21–08] makes it cell permeable. However, the CPP sequence interferes with the PIP binding to PCNA. ( B ) Design strategy: the PIP is coupled to a cell-penetrating peptide (in this case the TAT peptide) via a disulfide bridge; after transporting the PIP into the cell the disulfide bridge is reduced; the CPP accumulates mainly in the cytosol and nucleolus, while the PIP is free to reach and bind PCNA. ( C ) To visualize in live cells the separation of the PIP from CPP, we labeled each sequence with a different fluorescent dye. The TAT peptide labeled with TAMRA is coupled by a disulfide bridge to the PIP peptide labeled with FITC [R-TAT(-SS-)p21–08-F]. It can be seen that the free PIP labels the site of DNA damage, whereas the CPP distributes mainly in the cytosol and nucleolus. Experiments were repeated at least two times. Scale bar 5 μm.

Article Snippet: The sequences of the p21 peptide and the two highest PCNA binding affinity peptides p21–08 and PL screened are displayed. ( B ) Structures of the p21 peptides and the two PIPs, i.e., p21–08 and PL sequences obtained from molecular dynamics simulations are shown. ( C ) Images showing that these peptides ([F-p21], [F-p21–08], [F-PL]) after microinjection clearly co-localize with GFP-PCNA at replication sites. ( D ) When the peptides are added to the extracellular media they are not able to enter the cells.

Techniques: Marker, Expressing, Labeling, Sequencing, Binding Assay

Journal: Nucleus

Article Title: A novel cell permeable DNA replication and repair marker

doi: 10.4161/nucl.36290

Figure Lengend Snippet: Summary of peptides

Article Snippet: The sequences of the p21 peptide and the two highest PCNA binding affinity peptides p21–08 and PL screened are displayed. ( B ) Structures of the p21 peptides and the two PIPs, i.e., p21–08 and PL sequences obtained from molecular dynamics simulations are shown. ( C ) Images showing that these peptides ([F-p21], [F-p21–08], [F-PL]) after microinjection clearly co-localize with GFP-PCNA at replication sites. ( D ) When the peptides are added to the extracellular media they are not able to enter the cells.

Techniques: Sequencing

Journal: Bioconjugate chemistry

Article Title: Quantitative Assessment of Binding Affinities for Nanoparticles Targeted to Vulnerable Plaque

doi: 10.1021/acs.bioconjchem.5b00144

Figure Lengend Snippet: Physical Properties of SDIO–DO3A in Comparison to Those of DIO–DO3A

Article Snippet: The core sizes of DIO–DO3A and SDIO–DO3A were measured by transmission electron microscopy (TEM), using a Philips CM-12 operating at 80 kV.

Techniques: Comparison, Zeta Potential Analyzer

Journal: Molecular Biology and Evolution

Article Title: Understanding the Early Evolutionary Stages of a Tandem Drosophila melanogaster -Specific Gene Family: A Structural and Functional Population Study

doi: 10.1093/molbev/msaa109

Figure Lengend Snippet: Annotation of the Sdic region across seven populations of the DSPR panel. The most reliable organization of the region at 19C1 on the X chromosome in the ISO-1 is provided as a reference ( Clifton et al. 2017 ). The region is depicted from centromere (Cen) to telomere (Tel), including the flanking genes sw and AnxB10 (gray-filled arrows). Population names are color-coded based on the broad continental region where they were collected: green, Africa; red, Americas; and blue, Eurasia. The number of annotated Sdic copies in reference-quality genome assemblies ( Chakraborty et al. 2018 , ) is indicated in parentheses next to the name of the population. Sdic copies in the ISO-1 strain are named as reported ( Clifton et al. 2017 ). In the rest of populations, the copy identifiers are roman numerals according to their relative order from sw to AnxB10 . Sdic copies are color-coded, and a lower character ( a–m ) added to their identifier, both indicating the associated paratype. Three TE insertions (solid boxes) are shown, indicating both their size in kb and the location in relation to the gene structure (e, exon). One TE insertion is located within intronic sequence (A5_I), a common occurrence ( Chakraborty et al. 2018 ). In the other two cases, A7_III and B3_IV, the TE disrupts coding and 3′-UTR sequence, respectively. In the first case, the TE has possibly no functional consequence as a premature STOP codon resides upstream of the TE insertion; the apostrophe indicates an ancestral coding exon, which now situates outside of the predicted open reading frame.

Article Snippet: Primer design for Sdic took into consideration sequence differences with sw and AnxB10 to confidently survey solely Sdic expression, as well as perfect sequence conservation across copies and strains to prevent any copy or population bias.

Techniques: Sequencing, Functional Assay

Journal: Molecular Biology and Evolution

Article Title: Understanding the Early Evolutionary Stages of a Tandem Drosophila melanogaster -Specific Gene Family: A Structural and Functional Population Study

doi: 10.1093/molbev/msaa109

Figure Lengend Snippet: Global expression of the Sdic multigene family in whole-body males using qRT–PCR. ( A ) Sdic primers are shown relative to the Sdic transcriptional unit. See figure 3 A for details about the relationship of different parts of this transcriptional unit with the structure of the parental genes. Primers were designed upon examining the sequence of all the copies across all the strains of geographically diverse origin, plus ISO-1, making sure that there was no mismatch or gap. The upstream primer was designed spanning the intron between exons 1 and 2 of Sdic , with only 5 nt within exon 2, to prevent amplification of sw . ( B ) Fold change in expression of ten strains, including ISO-1 (value of 1 on the y axis), which was used as calibrator. Green, w 1118 and its synthetic derivatives carrying the duplication of the Sdic region (2T and 4M). Blue, strains of different geographical origin plus OR-R. Error bars, SEM. ( C ) Linear regression between CN and log2-fold change in expression for the two subsets of strains examined. Each dot represents the values obtained for each biological replicate included in the analysis. Determination coefficients ( r 2 ) and their corresponding P values are shown.

Article Snippet: Primer design for Sdic took into consideration sequence differences with sw and AnxB10 to confidently survey solely Sdic expression, as well as perfect sequence conservation across copies and strains to prevent any copy or population bias.

Techniques: Expressing, Quantitative RT-PCR, Sequencing, Amplification