A deep learning approach to photo–identification demonstrates high performance on two dozen cetacean species

We thank the countless individuals who collected and/or processed the nearly 85,000 images used in this study and those who assisted, particularly those who sorted these images from the millions that did not end up in the catalogues. Additionally, we thank the other Kaggle competitors who helped develop the ideas, models and data used here, particularly those who released their datasets to the public. The graduate assistantship for Philip T. Patton was funded by the NOAA Fisheries QUEST Fellowship. This paper represents HIMB and SOEST contribution numbers 1932 and 11679, respectively. The technical support and advanced computing resources from University of Hawaii Information Technology Services—Cyberinfrastructure, funded in part by the National Science Foundation CC* awards # 2201428 and # 2232862 are gratefully acknowledged. Every photo–identification image was collected under permits according to relevant national guidelines, regulation and legislation.Peer reviewedPublisher PD

Defining the Functional Domain of Programmed Cell Death 10 through Its Interactions with Phosphatidylinositol-3,4,5-Trisphosphate

Cerebral cavernous malformations (CCM) are vascular abnormalities of the central nervous system predisposing blood vessels to leakage, leading to hemorrhagic stroke. Three genes, Krit1 (CCM1), OSM (CCM2), and PDCD10 (CCM3) are involved in CCM development. PDCD10 binds specifically to PtdIns(3,4,5)P3 and OSM. Using threading analysis and multi-template modeling, we constructed a three-dimensional model of PDCD10. PDCD10 appears to be a six-helical-bundle protein formed by two heptad-repeat-hairpin structures (α1–3 and α4–6) sharing the closest 3D homology with the bacterial phosphate transporter, PhoU. We identified a stretch of five lysines forming an amphipathic helix, a potential PtdIns(3,4,5)P3 binding site, in the α5 helix. We generated a recombinant wild-type (WT) and three PDCD10 mutants that have two (Δ2KA), three (Δ3KA), and five (Δ5KA) K to A mutations. Δ2KA and Δ3KA mutants hypothetically lack binding residues to PtdIns(3,4,5)P3 at the beginning and the end of predicted helix, while Δ5KA completely lacks all predicted binding residues. The WT, Δ2KA, and Δ3KA mutants maintain their binding to PtdIns(3,4,5)P3. Only the Δ5KA abolishes binding to PtdIns(3,4,5)P3. Both Δ5KA and WT show similar secondary and tertiary structures; however, Δ5KA does not bind to OSM. When WT and Δ5KA are co-expressed with membrane-bound constitutively-active PI3 kinase (p110-CAAX), the majority of the WT is co-localized with p110-CAAX at the plasma membrane where PtdIns(3,4,5)P3 is presumably abundant. In contrast, the Δ5KA remains in the cytoplasm and is not present in the plasma membrane. Combining computational modeling and biological data, we propose that the CCM protein complex functions in the PI3K signaling pathway through the interaction between PDCD10 and PtdIns(3,4,5)P3

Proposed PtdIns(3,4,5)P₃ binding site and dimeric interface.

<p>A. Ribbon model of PDCD10 showing the proposed PIP binding Lys residues. B. Surface-potential model of PDCD10 showing the surface positive potential charge area (blue) and negative potential charge area (red). C. Superimposition of the ribbon model and the surface-potential model. Note the proposed PIP binding Lys residues. D. Superimposition of the ribbon model and the surface-potential model shows the potential dimeric interface. Note that the dimeric interaction surfaces are mutually exclusive to the PIP binding site.</p

Heptad repeats and the amphipathic helix.

<p>A. Sequence alignment of the N-terminal and the C-terminal three-helix bundles. B. Superimposition of the N-terminal and C-terminal three-helix bundles shows that these two heptad-repeat-hairpin structures are related in the three dimensional structures. C. A diagram and primary sequence of the proposed amphipathic helix and the PIP binding site.</p

PDCD10 interactions with phospholipids and OSM.

<p>A. The Membrane Lipid Array shows that PDCD10 binds exclusively to phosphotidylserine with weak binding. B. PIP Arrays show the relative PIP binding affinity for the WT and three mutant PDCD10 proteins. C. Pull-down experiments, using recombinant purified WT and Δ5KA PDCD10 coupled to sepharose beads to pull-down FLAG-tagged overexpressed OSM and Krit1, showing that only the WT binds to the OSM-Krit1 complex.</p

Co-localization of PDCD10 and p110-CAAX.

<p>Overexpression of the WT and Δ5KA PDCD10 together with p110-CAAX shows that WT PDCD10 and p110-CAAX colocalize to the membrane while the Δ5KA stays only in the cytoplasm. A. WT in Mcherry. B. Δ5KA in CFP. C. p110-CAAX in FITC. D. A composite picture of WT (Mcherry) and p110-CAAX (FITC). E. A composite picture of Δ5KA (CFP) and p110-CAAX (FITC). F. A composite picture of WT (Mcherry) and Δ5KA (CFP). G. A composite picture of WT (Mcherry), p110-CAAX (FITC), and Δ5KA (CFP). H. A composite picture of Δ5KA (CFP) and p110-CAAX (FITC). A light cyan mask was used to enhance the visualization of Δ5KA localization.</p

PDCD10 Model.

<p>A. Multiple sequence alignment of mouse, human, and zebra fish PDCD10 shows a highly conserved primary structure. B/C. A three dimensional model for PDCD10 shows a double heptad-repeat-hairpin structure.</p

Determination of quaternary structure of wild-type and mutant Δ5KA ccm3 in solution using size exclusion chromatrography/multi angle laser light scattering (SEC-MAL).

<p>Determination of quaternary structure of wild-type and mutant Δ5KA ccm3 in solution using size exclusion chromatrography/multi angle laser light scattering (SEC-MAL).</p

Proposed PDCD10 signaling model.

<p>Proposed PDCD10 signaling model.</p

Circular dichroism and fraction denaturation.

<p>A. CD spectra of the WT and Δ5KA PDCD10 showing identical CD spectra. B. Fraction denaturation experiments show the lower Tm of Δ5KA compared to the WT.</p