RESUMO
Penaeid shrimp embryos undergo holoblastic division, gastrulation by invagination, and hatching as a nauplius larva. Posterior segments form and differentiate during larval development. Hedgehog (Hh) pathway genes from penaeid shrimp and other pancrustaceans were identified by in silico analysis of genomes and transcriptomes, and mapped onto a recent pancrustacean phylogeny to determine patterns of intron gains and losses. Penaeus vannamei, P. japonicus, and P. monodon Hh proteins were encoded by four exons. Amphipod, isopod, and ostracod hh were also encoded by four exons, but hh from other arthropod groups contained three conserved exons. The novel hh intron is hypothesized to have arisen independently in the malacostracan ancestor and Ostracoda by a transposon insertion. Shared patterns of ptc, smo, and ci exon structure were found for Malacostraca, Branchiopoda + Hexapoda, Hexanauplia (Thecostraca + Copepoda), Multicrustacea (Thecostraca + Copepoda + Malacostraca), and Pancrustacea minus Oligostraca. mRNA expression of P. vannamei of hh, ptc, and ci from developmental transcriptomes of zygotes through postlarvae showed low expression from zygote to gastrula, which increased at limb bud, peaked at unhatched nauplius, and declined in nauplius and later larval stages. smo expression was found in zygotes, peaked in gastrula, and declined in limb bud and later stages. These results are consistent with a role for Hh signaling during segmentation in penaeid shrimp.
Assuntos
Artrópodes , Penaeidae , Animais , Proteínas Hedgehog/genética , Insetos/genética , Larva/genética , Penaeidae/genética , FilogeniaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Programmed cell death ligand-1 (PD-L1) expression levels in patient tumor samples have proven clinical utility across various cancer types. Several independently developed PD-L1 immunohistochemical (IHC) predictive assays are commercially available. Published studies using the VENTANA PD-L1 (SP263) Assay, VENTANA PD-L1 (SP142) Assay, Dako PD-L1 IHC 22C3 pharmDx assay, Dako PD-L1 IHC 28-8 pharmDx assay, and laboratory-developed tests utilizing the E1L3N antibody (Cell Signaling Technology), have demonstrated differing levels of PD-L1 staining between assays, resulting in conjecture as to whether antibody-binding epitopes could be responsible for discordance between assays. Therefore, to understand the performance of different PD-L1 predictive immunohistochemistry assays, we aimed to distinguish the epitopes within the PD-L1 protein responsible for antibody binding. The sites at which antibody clones SP263, SP142, 22C3, 28-8, and E1L3N bind to recombinant PD-L1 were assessed using several methods, including conformational peptide array, surface plasmon resonance, and/or hydrogen/deuterium exchange mass spectrometry. Putative binding sites were confirmed by site-directed mutagenesis of PD-L1, followed by western blotting and immunohistochemical analysis of cell lines expressing mutant constructs. Our results demonstrate that clones SP263 and SP142 bind to an identical epitope in the cytoplasmic domain at the extreme C-terminus of PD-L1, distinct from 22C3 and 28-8. Using mutated PD-L1 constructs, an additional clone, E1L3N, was also found to bind to the cytoplasmic domain of PD-L1. The E1L3N binding epitope overlaps considerably with the SP263/SP142 binding site but is not identical. Clones 22C3 and 28-8 have binding profiles in the extracellular domain of PD-L1, which differ from one another. Despite identifying epitope binding variance among antibodies, evidence indicates that only the SP142 assay generates significantly discordant immunohistochemical staining, which can be resolved by altering the assay protocol. Therefore, inter-assay discordances are more likely attributable to tumor heterogeneity, assay, or platform variables rather than antibody epitope.