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Data from: Advanced characterization of DNA molecules in rAAV vector preparations by single-stranded virus next-generation sequencing
Recent successful clinical trials with recombinant adeno-associated viral vectors (rAAVs) have led to a renewed interest in gene therapy. However, despite extensive developments to improve vector-manufacturing processes, undesirable DNA contaminants in rAAV preparations remain a major safety concern. Indeed, the presence of DNA fragments containing antibiotic resistance genes, wild-type AAV, and packaging cell genomes has been found in previous studies using quantitative polymerase chain reaction (qPCR) analyses. However, because qPCR only provides a partial view of the DNA molecules in rAAV preparations, we developed a method based on next-generation sequencing (NGS) to extensively characterize single-stranded DNA virus preparations (SSV-Seq). In order to validate SSV-Seq, we analyzed three rAAV vector preparations produced by transient transfection of mammalian cells. Our data were consistent with qPCR results and showed a quasi-random distribution of contaminants originating from the packaging cells genome. Finally, we found single-nucleotide variants (SNVs) along the vector genome but no evidence of large deletions. Altogether, SSV-Seq could provide a characterization of DNA contaminants and a map of the rAAV genome with unprecedented resolution and exhaustiveness. We expect SSV-Seq to pave the way for a new generation of quality controls, guiding process development toward rAAV preparations of higher potency and with improved safety profiles.
FIGURES 1415 in Systematics and biogeography of the spider genus Mallinella Strand, 1906, with descriptions of new species and new genera from Southeast Asia (Araneae, Zodariidae) 3369
FIGURES 1415. Distribution range of Mallinella species.
Annotated DNA Double Strand Break Ionizing Radiation-Induced Foci (gH2AX 53BP1) Confocal Microscopy, pt. 1
<p><strong>Summary</strong></p> <p>Dataset of confocal microscopy data of cells exposed to gamma-irradiation and immunostained with gH2AX and 53BP1.</p> <ul> <li>Nuclei segmentation Head and neck primocultures immunostained with gH2AX/53BP1, Training/Testing/Validation dataset (nucleus_segmentation.zip)</li> <li>IRIF Foci: Head and neck primocultures immunostained with gH2AX/53BP1, Training/Testing/Validation dataset. (foci_detection.zip)</li> <li>Cell lines: U-87 and NHDF Cells exposed to 0.5-8 Gy 30 min and 8h post irradiation - confocal microscopy data of gH2AX/53BP1/DAPI annotated for gH2AX, 53BP1 and colocalized foci separately (cell_lines_U87.zip and NHDF.zip part 2 of dataset, available at <a href="https://dx.doi.org/10.5281/zenodo.5549971">10.5281/zenodo.5549971</a>). </li> <li><strong>Code</strong>: the code is available at <a href="https://github.com/tomasvicar/DeepFoci">https://github.com/tomasvicar/DeepFoci</a></li> <li><strong>Preprint: </strong>Vicar et al, DeepFoci: Deep Learning-Based Algorithm for Fast Automatic Analysis of DNA Double Strand Break Ionizing Radiation-Induced Foci, <a href="https://doi.org/10.1101/2020.10.07.321927">10.1101/2020.10.07.321927</a></li> <li><strong>Publication: </strong>Vicar et al, TBA</li> </ul> <p><strong>Materials and methods</strong></p> <p><em>Dataset</em></p> <p>Following cells were used:</p> <p>1) The training/validation/testing datasets was based on patient-derived primary cell cultures prepared from spinocellular tumors and morphologically normal tissues adjacent to the tumor taken from patients suffering from head and neck cancer. The dataset was divided into two subsets: one for training, validation and testing the nucleus segmentation (237/10/30 fields of view (FOVs), respectively) and one for training, validation and testing the focus segmentation (239/60/100 FOVs). The dataset consisted of several cell types: a) tumor cells, b) tumor-associated fibroblasts, and c) cells from morphologically normal tissues. All cell types were fixed at different periods of time (0 (non-irradiated control), 0.5, 8 or 24 h PI) after exposure to 2 Gy of gamma-rays. The representation of cells in two subsets with respect to the cell type and post-irradiation time (i.e., DSB repair duration) was random.</p> <p>2) The evaluation dataset was used to assess the robustness of segmentation procedures. It was composed of multiple types of differently treated cells in order to represent a highly challenging dataset maximally reflecting high biological and technical variability between samples, as it may appear in research or clinical practice. The dataset contained a) mesenchymal NHDF fibroblasts coming from a standard permanent cell line, b) radioresistant U-87 glioblastoma cells coming from a standard permanent cell line, c) tumor cells (CD90-) and tumor-associated fibroblasts (CD90+) prepared as a primary culture from a spinocellular tumors of patients (different from dataset 1) suffering from a head and neck cancer, and d) cells prepared as primary cultures from morphologically normal tissue adjacent to tumors of involved head and neck cancer patients. NHDF and U-87 cells received 0.5, 0, 1, 2, 4 and 8 Gy of gamma-rays and were fixed at 30 min and 8 h post-irradiation, while the primary cultures were only exposed to the dose of 2 Gy (for a limited amount of the cell material) and fixed at 0 (non-irradiated control), 0.5, 8 or 24 h post-irradiation times.</p> <p><em>Gamma irradiation</em></p> <p>The cells were irradiated at the Institute of Biophysics, Czech Academy of Sciences, Brno, Czech Republic in a following schemes: (a) patient-derived primoculture was irradiated with a single dose of 2 Gy (D = 1 Gy/min) of gamma-rays (60Co, Chisostat, Chirana, CR) , (b) U-87 and NHDF cells were irradiated with doses 0.5-8 Gy (D = 1 Gy/min). Cells were irradiated in RPMI 1640 medium (37 °C, normal atmosphere). Confocal microscopy of gammaH2AX and 53BP1 foci immunodetection was consequently performed.</p> <p><em>Fluorescent staining</em></p> <p>DNA double strand breaks (DSBs) were quantified in different periods of time post-irradiation (30 min, 8h and 24h post irradiation) by means of $\gamma$H2AX and 53BP1 foci immunodetection combined with confocal microscopy. For details see \cite{falk2007chromatin}.</p> <p><em>Confocal microscopy</em></p> <p>The microscopy of samples was performed at the Institute of Biophysics, Czech Academy of Sciences, Brno, Czech Republic. Leica DM RXA microscope (equipped with DMSTC motorized stage, Piezzo z-movement, MicroMax CCD camera, CSU-10 confocal unit and 488, 562, and 714 nm laser diodes with AOTF) was used for acquiring detailed cell images (100× oil immersion Plan Fluotar lens, NA 1.3). Total 50 Z slices was captured with Z step size 0.3 μm.</p> <p><strong>File description</strong></p> <p>all files are compressed hyperstack tiffs (50 Z slices and 3 fluorescent channels, XYCZ order), 100x magnification</p> <ul> <li>foci_detection.zip: IRIF Foci: Head and neck primocultures immunostained with gH2AX/53BP1, Training/Testing/Validation dataset: FOVs 240/100/120 files for training, testing, and validation, organisation: <ul> <li>data_001.tif – 3channel Z.stack tiff</li> <li>mask_001.tif – respective Z stack mask with single points per IRIF focus (manual annotation, training and testing subsets only)</li> <li>data_description.xlsx – description of sample type (2Gy post irradiation times and characteristics of primary culture of squamous cell cancer of patients)</li> <li>data_001_pos.csv – manual annotation of gH2AX/53BPI IRIF foci by two experts – cordinates file (in 2D, validation only)</li> </ul> </li> <li>nucleus_segmentation.zip Nuclei segmentation Head and neck primocultures immunostained with gH2AX/53BP1, FOVs 237 Training/ 30 Testing/ 10 Validation dataset <ul> <li>data_001.tif – 3channel Z.stack tiff</li> <li>mask_001.tif – respective Z stack mask with manually annotated nucleus mask (manual annotation, training and testing subsets only)</li> <li>data_description.xlsx – description of sample type (2Gy post irradiation times and characteristics of primary culture of squamous cell cancer of patients)</li> </ul> </li> <li>U87.zip and NHDF.zip: Annotated gH2AX/53BP1 foci in cell lines exposed to increasing dose, annotations performed for gH2AX, 53BP1 and colocalized focus separatelly. 679 annotated FOVs for both cell lines (part 2 of dataset, available at <a href="https://dx.doi.org/10.5281/zenodo.5549971">10.5281/zenodo.5549971</a>). <ul> <li>control.png - RGB control figure showing merge and annotated overlay</li> <li>data_53BP1.tif - TIFF Z-stack, confocal microcopy, 53BP1 channel</li> <li>data_DAPI.tif - TIFF Z-stack, confocal microcopy, DAPI channel</li> <li>data_gH2AX.tif - TIFF Z-stack, confocal microcopy, gH2AX channel</li> <li>labels.json - foci labels for individual channel</li> <li>mask.tif - generated Z-stack of nucleus mask</li> </ul> </li> </ul>
FIGURES 25–27 in Phlegra Simon, 1876, Phintella Strand 1906 and Yamangalea Maddison, 2009 (Arachnida: Araneae: Salticidae) — new species and new generic records for Australia
FIGURES 25–27. Yamangalea lubinae sp. nov. Paratype: 25–26 palpal organ; 27 chelicera.
Figure 9 from: Huang Y, Wang C, Peng X-J (2015) Five new species of Phintella Strand, 1906 (Araneae, Salticidae) from the Wuling Mountains, China. ZooKeys 514: 25-42. https://doi.org/10.3897/zookeys.514.9159
Figure 9 - Phintella pulcherrima sp. n., A female body, dorsal view B epigyne, ventral view C vulva, dorsal view. Scale bars: 1.0 mm (A); 0.1 mm (B–C).
Figure 8 from: Huang Y, Wang C, Peng X-J (2015) Five new species of Phintella Strand, 1906 (Araneae, Salticidae) from the Wuling Mountains, China. ZooKeys 514: 25-42. https://doi.org/10.3897/zookeys.514.9159
Figure 8 - Phintella pulcherrima sp. n., A male body, dorsal view B male palp, ventral view C male palp, retrolateral view. Scale bars: 1.0 mm (A); 0.1 mm (B–C).
Figure 7 from: Huang Y, Wang C, Peng X-J (2015) Five new species of Phintella Strand, 1906 (Araneae, Salticidae) from the Wuling Mountains, China. ZooKeys 514: 25-42. https://doi.org/10.3897/zookeys.514.9159
Figure 7 - Phintella panda sp. n., A epigynum, ventral view B vulva, dorsal view. AM atrium margin BP basal plate CD copulatory ducts FD fertilization duct S spermathecae. Scale bars: 0.1 mm (A–B).
Figure 4 from: Huang Y, Wang C, Peng X-J (2015) Five new species of Phintella Strand, 1906 (Araneae, Salticidae) from the Wuling Mountains, China. ZooKeys 514: 25-42. https://doi.org/10.3897/zookeys.514.9159
Figure 4 - Phintella levii sp. n., A female body, dorsal view B epigyne, ventral view C vulva, dorsal view. Scale bars: 1.0 mm (A); 0.1 mm (B–C).
Figure 6 from: Huang Y, Wang C, Peng X-J (2015) Five new species of Phintella Strand, 1906 (Araneae, Salticidae) from the Wuling Mountains, China. ZooKeys 514: 25-42. https://doi.org/10.3897/zookeys.514.9159
Figure 6 - Phintella panda sp. n., A female body, dorsal view B epigyne, ventral view C vulva, dorsal view. Scale bars: 1.0 mm (A); 0.1 mm (B–C).
Figure 5 from: Huang Y, Wang C, Peng X-J (2015) Five new species of Phintella Strand, 1906 (Araneae, Salticidae) from the Wuling Mountains, China. ZooKeys 514: 25-42. https://doi.org/10.3897/zookeys.514.9159
Figure 5 - Phintella levii sp. n., A epigynum, ventral view B vulva, dorsal view. AM atrium margin BP basal plate CD copulatory duct FD fertilization duct PS poriform structure S spermathecae. Scale bars: 0.1 mm (A–B).
Figure 3 from: Huang Y, Wang C, Peng X-J (2015) Five new species of Phintella Strand, 1906 (Araneae, Salticidae) from the Wuling Mountains, China. ZooKeys 514: 25-42. https://doi.org/10.3897/zookeys.514.9159
Figure 3 - Phintella arcuata sp. n., A male palp, ventral view B male palp, retrolateral view C left chelicerae of male, posterior view D epigyne, ventral view E vulva, dorsal view. AM atrium margin BP basal plate CD copulatory duct E embolus FD fertilization duct LP lamellar process PL posterior lobe PS poriform structure RTA retrolateral tibial apophysis TB tegulum bump TSDA terminal sperm duct angle S spermathecae. Scale bars: 0.1 mm (A–E).
Figure 2 from: Huang Y, Wang C, Peng X-J (2015) Five new species of Phintella Strand, 1906 (Araneae, Salticidae) from the Wuling Mountains, China. ZooKeys 514: 25-42. https://doi.org/10.3897/zookeys.514.9159
Figure 2 - Phintella arcuata sp. n., A female body, dorsal view B epigyne, ventral view C vulva, dorsal view. Scale bars: 1.0 mm (A); 0.1 mm (B–C).
Figure 13 from: Huang Y, Wang C, Peng X-J (2015) Five new species of Phintella Strand, 1906 (Araneae, Salticidae) from the Wuling Mountains, China. ZooKeys 514: 25-42. https://doi.org/10.3897/zookeys.514.9159
Figure 13 - Distribution records of all new species. A ● Phintella arcuata; ▲ Phintella pulcherrima B Phintella levii C Phintella panda D Phintella wulingensis.
Figure 10 from: Huang Y, Wang C, Peng X-J (2015) Five new species of Phintella Strand, 1906 (Araneae, Salticidae) from the Wuling Mountains, China. ZooKeys 514: 25-42. https://doi.org/10.3897/zookeys.514.9159
Figure 10 - Phintella pulcherrima sp. n., A male palp, ventral view B male palp, retrolateral view C epigyne, ventral view D vulva, dorsal view. Scale bars: 0.1 mm (A–D). AM atrium margin BP basal plate CD copulatory ducts E embolus FD fertilization ducts LP lamellar process PL posterior lobe RTA retrolateral tibial apophysis TB tegulum bump; S spermathecae.
Figure 11 from: Huang Y, Wang C, Peng X-J (2015) Five new species of Phintella Strand, 1906 (Araneae, Salticidae) from the Wuling Mountains, China. ZooKeys 514: 25-42. https://doi.org/10.3897/zookeys.514.9159
Figure 11 - Phintella wulingensis sp. n., A female body, dorsal view B epigyne, ventral view C vulva, dorsal view. Scale bars: 1.0 mm (A); 0.1 mm (B–C).
Figure 12 from: Huang Y, Wang C, Peng X-J (2015) Five new species of Phintella Strand, 1906 (Araneae, Salticidae) from the Wuling Mountains, China. ZooKeys 514: 25-42. https://doi.org/10.3897/zookeys.514.9159
Figure 12 - Phintella wulingensis sp. n., A epigynum, ventral view B vulva, dorsal view. AM atrium margin BP basal plate CD copulatory duct CO copulatory opening FD fertilization duct S spermathecae. Scale bars: 0.1 mm (A–B).
Figure 1 from: Huang Y, Wang C, Peng X-J (2015) Five new species of Phintella Strand, 1906 (Araneae, Salticidae) from the Wuling Mountains, China. ZooKeys 514: 25-42. https://doi.org/10.3897/zookeys.514.9159
Figure 1 - Phintella arcuata sp. n., A male body, dorsal view B left chelicerae of male, posterior view C male palp, ventral view D male palp, retrolateral view. Scale bars: 1.0 mm (A); 0.1 mm (B); 0.2 mm (C–D).
Figure 4 from: Peris-Felipo FJ, Yari Z, van Achterberg C, Rakhshani E, Belokobylskij SA (2016) Review of species of the genus Adelurola Strand, 1928, with a key to species (Hymenoptera, Braconidae, Alysiinae). ZooKeys 566: 13-30. https://doi.org/10.3897/zookeys.566.6684
Figure 4 - Adelurola florimela (Haliday) (female). A Habitus, lateral view B Head (lateral view) and mandible C Face, front view D Mesosoma, lateral view E Metasoma and ovipositor, lateral view F First metasomal tergite G Fore and hind wings.
Figure 3 from: Peris-Felipo FJ, Yari Z, van Achterberg C, Rakhshani E, Belokobylskij SA (2016) Review of species of the genus Adelurola Strand, 1928, with a key to species (Hymenoptera, Braconidae, Alysiinae). ZooKeys 566: 13-30. https://doi.org/10.3897/zookeys.566.6684
Figure 3 - Adelurola asiatica Telenga (female). A Habitus, lateral view B Face, front view C Mandible D Mesosoma, lateral view E Metasoma and ovipositor, lateral view F First metasomal tergite G Fore and hind wings.
Figure 2 from: Peris-Felipo FJ, Yari Z, van Achterberg C, Rakhshani E, Belokobylskij SA (2016) Review of species of the genus Adelurola Strand, 1928, with a key to species (Hymenoptera, Braconidae, Alysiinae). ZooKeys 566: 13-30. https://doi.org/10.3897/zookeys.566.6684
Figure 2 - Adelurola amplidens (Fischer) (female). A Habitus, lateral view B Head (lateral view) and mandible C Face, front view D Mesosoma, lateral view E Hind femur, metasoma and ovipositor, lateral view F First metasomal tergite G Fore and hind wings.
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Allen Brain Atlas
Allen Brain Atlas is an Allen Institute collection of brain map atlases, datasets, APIs, and analysis tools covering mouse, human, and non-human primate brain resources.
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DANDI Archive for NWB datasets
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International Brain Laboratory public data
The International Brain Laboratory public data releases expose standardized mouse decision-making experiments, including Neuropixels recordings, widefield calcium imaging, behavior, and session metadata accessed through the ONE API.
OpenNeuro
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