G protein-coupled receptors (GPCRs) play a crucial role in cell function by transducing signals from the extracellular environment to the inside of the cell. They mediate the effects of various stimuli, including hormones, neurotransmitters, ions, photons, food tastants and odorants, and are renowned drug targets. Advancements in structural biology techniques, including X-ray crystallography and cryo-electron microscopy (cryo-EM), have driven the elucidation of an increasing number of GPCR structures. These structures reveal novel features that shed light on receptor activation, dimerization and oligomerization, dichotomy between orthosteric and allosteric modulation, and the intricate interactions underlying signal transduction, providing insights into diverse ligand-binding modes and signalling pathways. However, a substantial portion of the GPCR repertoire and their activation states remain structurally unexplored. Future efforts should prioritize capturing the full structural diversity of GPCRs across multiple dimensions. To do so, the integration of structural biology with biophysical and computational techniques will be essential. We describe in this review the progress of nuclear magnetic resonance (NMR) to examine GPCR plasticity and conformational dynamics, of atomic force microscopy (AFM) to explore the spatial-temporal dynamics and kinetic aspects of GPCRs, and the recent breakthroughs in artificial intelligence for protein structure prediction to characterize the structures of the entire GPCRome. In summary, the journey through GPCR structural biology provided in this review illustrates how far we have come in decoding these essential proteins architecture and function. Looking ahead, integrating cutting-edge biophysics and computational tools offers a path to navigating the GPCR structural landscape, ultimately advancing GPCR-based applications. LINKED ARTICLES: This article is part of a themed issue Complexity of GPCR Modulation and Signaling (ERNST). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v182.14/issuetoc.

• MeSH
• Protein Conformation MeSH
• Humans MeSH
• Receptors, G-Protein-Coupled * chemistry   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

Dystonia is a movement disorder characterized by genetic and clinical heterogeneity. A recurring p.(Glu303del)-deletion in TOR1A is a well-established cause for DYT-TOR1A (DYT1), an autosomal dominant early-onset isolated dystonia. TOR1A encodes TorsinA, an AAA + ATPase located in the nuclear envelope. By whole exome analyses of a family with a novel dystonia-hemichorea-/hemiballism phenotype, we identified a TOR1AIP2 NM_001199260.2 c.1234A > G p.(Arg412Gly) variant. The variant is very rare in databases and was absent from whole exome data from >1000 dystonia patients. TOR1AIP2 encodes LULL1, a transmembrane protein that activates TorsinA, and correct interaction between TorsinA and LULL1 is essential for proper nuclear envelope architecture. The p.(Arg412Gly) variant disrupts the binding interface between TorsinA and LULL1 around p.Arg412; this same interface is also impaired in DYT1. Functional analyses via a co-purification assay revealed that interaction between TorsinA-LULL1Arg412Gly is weaker than the wild-type interaction, and that it resembles the situation in DYT1 (TorsinAΔE303-LULL1). A second family with milder dystonia, hemichorea, and stereotypic leg flexion during gait and a TOR1AIP2 p.(Gln338His) variant was identified. The clinical phenotype of both families shared proximal arm movements, and flutter in facial musculature. Expressivity of the movement disorder symptoms was variable. Several proteins in the nuclear envelope have been implicated in various forms of neurodevelopmental disorders with dystonia. Taken together, our findings suggest TOR1AIP2 as a new candidate gene implicated in a complex hereditary movement disorder with dystonia and hemichorea/hemiballism.

• MeSH
• Chorea * genetics   MeSH
• Adult MeSH
• Dyskinesias * genetics   MeSH
• Dystonic Disorders * genetics   MeSH
• Dystonia * genetics   MeSH
• Middle Aged MeSH
• Humans MeSH
• Molecular Chaperones * genetics   MeSH
• Pedigree MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

AIMS: Benign tumours of the rete testis include mostly cystadenomas and adenomas. A subset with tubular or tubulopapillary architecture shows morphological similarities to Sertoli cell tumours; these neoplasms were previously termed "Sertoliform cystadenomas of the rete testis". In the most recent WHO classification, they have been interpreted as Sertoli cell tumours, not otherwise specified (NOS), with pure intra-rete growth, and therefore excluded as an entity. The remaining cystadenomas of the rete testis vaguely resemble tumours of Mullerian origin arising in the ovaries. In this study we analyse benign tumours of the rete testis, including a subset with Sertoliform features. METHODS AND RESULTS: Benign neoplasms of the rete testis were identified through query of consultation and institutional files. Clinicopathologic data were collected, and available slides were reviewed. Cases were assessed using IHC and three separate DNA sequencing panels. Eleven tumours from patients 32-78 years old were evaluated. Four were classified as Sertoliform adenomas/cystadenomas, displaying tubulo-papillary or tubular/trabecular architecture; all of them were PAX8-positive and lacked nuclear beta-catenin expression. The remaining seven tumours were benign cystadenomas NOS. Genomic analysis was performed successfully in 10/11 tumours (including all Sertoliform adenomas/cystadenomas) and revealed no pathogenic variants in CTNNB1, KRAS, or BRAF. CONCLUSION: Sertoliform cystadenomas of the rete testis differ from Sertoli cell tumours NOS, as evidenced by the absence of molecular markers characteristic of Sertoli cell tumours. The remaining benign cystadenomas lack molecular alterations seen in Mullerian tumors of the ovaries.

• MeSH
• Cystadenoma * pathology   genetics   MeSH
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Sertoli Cell Tumor * pathology   diagnosis   MeSH
• Biomarkers, Tumor analysis   MeSH
• Rete Testis * pathology   MeSH
• Aged MeSH
• Testicular Neoplasms * pathology   genetics   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Publication type
• Journal Article MeSH

The RNA content is crucial for the formation of nuclear compartments, such as nuclear speckles and nucleoli. Phosphatidylinositol 4,5-bisphosphate (PIP2) is found in nuclear speckles, nucleoli, and nuclear lipid islets and is involved in RNA polymerase I/II transcription. Intriguingly, the nuclear localization of PIP2 was also shown to be RNA-dependent. We therefore investigated whether PIP2 and RNA cooperate in the establishment of nuclear architecture. In this study, we unveiled the RNA-dependent PIP2-associated (RDPA) nuclear proteome in human cells by mass spectrometry. We found that intrinsically disordered regions (IDRs) with polybasic PIP2-binding K/R motifs are prevalent features of RDPA proteins. Moreover, these IDRs of RDPA proteins exhibit enrichment for phosphorylation, acetylation, and ubiquitination sites. Our results show for the first time that the RDPA protein Bromodomain-containing protein 4 (BRD4) associates with PIP2 in the RNA-dependent manner via electrostatic interactions, and that altered PIP2 levels affect the number of nuclear foci of BRD4 protein. Thus, we propose that PIP2 spatiotemporally orchestrates nuclear processes through association with RNA and RDPA proteins and affects their ability to form foci presumably via phase separation. This suggests the pivotal role of PIP2 in the establishment of a functional nuclear architecture competent for gene expression.

• MeSH
• Cell Nucleus * metabolism   genetics   MeSH
• Phosphatidylinositol 4,5-Diphosphate * metabolism   MeSH
• Phosphorylation MeSH
• Nuclear Proteins * metabolism   genetics   MeSH
• Humans MeSH
• Cell Cycle Proteins metabolism   genetics   MeSH
• Bromodomain Containing Proteins MeSH
• RNA metabolism   genetics   MeSH
• Transcription Factors * metabolism   genetics   MeSH
• Protein Binding MeSH
• Intrinsically Disordered Proteins * metabolism   genetics   chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

We report a very unusual case of melanocytic neoplasm appearing clinically as a 0.5-cm dome-shaped pigmented papule on the chest of a 63-year-old man. Microscopically, it was an asymmetric, entirely dermally based neoplasm characterized by a multinodular, vaguely plexiform architecture composed of moderately pleomorphic spindled melanocytes with ample, dusty pigmented cytoplasm and scattered multinucleated cells. The tumor cells were strongly positive for Melan-A, HMB45, S100, and PRAME, whereas p16 showed diffuse nuclear loss. β-catenin presented a strong and diffuse cytoplasmic staining, while nuclei were negative. Despite an increased cellularity, mitotic count was low (1/mm 2 ). Fluorescence in situ hybridization revealed no copy number alteration in melanoma-related genes ( CDKN2A, MYB, MYC, CCND1 and RREB1 ). DNA and RNA sequencing identified KIT c.2458G>T and APC c.6709C>T mutations. No further genetic alteration was detected including TERT-promoter (TERT-p ) hot-spot mutation. A re-excision was performed. A sentinel lymph node biopsy was negative. Clinical investigations revealed no extracutaneous involvement. The patient is disease-free after a follow-up period of 8 months. Given the peculiar morphologic and molecular findings, we hypothesize the lesion may represent a novel subtype of an intermediate grade melanocytic tumor (melanocytoma).

• MeSH
• Antigens, Neoplasm MeSH
• Sentinel Lymph Node Biopsy MeSH
• In Situ Hybridization, Fluorescence MeSH
• Middle Aged MeSH
• Humans MeSH
• Melanocytes pathology   MeSH
• Melanoma * pathology   MeSH
• Mutation MeSH
• Skin Neoplasms * pathology   MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Publication type
• Journal Article MeSH
• Case Reports MeSH

Uterine tumor resembling ovarian sex cord tumor (UTROSCT) is a rare tumor of uncertain lineage and low malignant potential. Most tumors behave in a benign manner, but a subset of UTROSCT exhibit an aggressive clinical course with recurrences and metastases. The recurrent molecular alterations in UTROSCT mostly represent gene fusions involving NCOA1-3. We performed a comprehensive clinicopathological, morphologic, immunohistochemical, and molecular analysis on a cohort of 35 UTROSCT. The tumors exhibited various architectural patterns (diffuse, corded/trabecular, tubular, sertoliform, fascicular, whorled, nested, microfollicular, and pseudoglandular), often in combination. The immunohistochemical analysis confirmed the polyphenotypic immunoprofile, often with coexpression of sex cord-stromal, smooth muscle, and epithelial markers, as well as hormone receptors. Next-generation sequencing RNA analysis revealed recurrent NCOA1-3 gene fusions in 22/32 analyzed cases (69%), including ESR1::NCOA3 (11/22), GREB1::NCOA2 (7/22), ESR1::NCOA2 (3/22), and GREB1::NCOA1 (1/22). Tumor mutation burden was low in all cases. The fusion-positive cases exhibited statistically significant association with whorled architecture, conversely necrosis was associated with fusion-negative status. We did not find a significant relationship between any architectural pattern and GREB1 alterations, but the NCOA2-altered tumors were associated with pseudoglandular architecture. The GREB1-altered cases occurred in older patients and tended to be more often intramural masses compared with ESR1-altered cases. On the contrary, the ESR1-altered cases presented more often like submucosal or polypoid tumors. Two tumors exhibited aggressive behavior with recurrent disease. Both of these cases harbored a GREB1::NCOA2 fusion. Unsupervised hierarchical cluster analysis of our cohort revealed 2 main clusters. The tumors with GREB1 or NCOA2 fusion cluster together, suggesting that there are underlying molecular differences between these cases and cases with ESR1::NCOA3 fusion or without fusion. Our findings contribute to the growing knowledge about a rare neoplasm with currently uncertain biological behavior.

• MeSH
• Adult MeSH
• Sex Cord-Gonadal Stromal Tumors * genetics   pathology   MeSH
• Immunohistochemistry * MeSH
• Nuclear Receptor Coactivator 2 genetics   MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Biomarkers, Tumor * genetics   analysis   MeSH
• Uterine Neoplasms * genetics   pathology   MeSH
• Ovarian Neoplasms genetics   pathology   MeSH
• Aged MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

Adenoid cystic carcinoma (AdCC) is one of the most common salivary gland malignancies and occurs in all major and minor salivary gland and seromucous gland sites. AdCCs of salivary gland origin have long been categorized as fusion-defined carcinomas owing to the almost consistent presence of fusion genes MYB::NFIB , or less commonly MYBL1::NFIB. We collected a cohort of 95 cases of AdCC, which were largely characterized by canonical fusions MYB::NFIB (49 cases) or MYBL1::NFIB (9 cases). In additional 11 cases of AdCC, rearrangements in MYB or NFIB genes were detected by FISH. In addition, NGS revealed novel noncanonical fusion transcripts EWSR1::MYB ; ACTB::MYB; ESRRG::DNM3, MYB::TULP4 , and ACTN4::MYB , each of them in 1 case. The tumors that showed noncanonical fusions had features of metatypical AdCC with a diverse architecture, lobulated multinodular growth pattern, and hypercellular peripheral palisading of nuclei (2 cases), tubular hypereosinophilia (2 cases), and pale eosinophilic to vacuolated (bubbly) cytoplasm (3 cases). Our study documented 3 cases of AdCC of salivary glands harboring novel gene fusions TULP4::MYB , ACTN4::MYB , and ACTB::MYB , in 1 case each, which have not been described before. A rare EWSR1::MYB fusion was detected in 1 case. Moreover, 1 case of sinonasal metatypical AdCC showed EWSR1 rearrangement detected by FISH. Also, 1 case with an ESRRG::DNM3 fusion of unknown significance is described in this study. These discoveries illustrate how broad molecular profiling will expand understanding of changes in known entities.

• MeSH
• Carcinoma, Adenoid Cystic * genetics   pathology   MeSH
• Adult MeSH
• Gene Fusion MeSH
• Oncogene Proteins, Fusion * genetics   MeSH
• Genetic Predisposition to Disease MeSH
• Gene Rearrangement MeSH
• In Situ Hybridization, Fluorescence MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Biomarkers, Tumor * genetics   MeSH
• Salivary Gland Neoplasms * genetics   pathology   MeSH
• RNA-Binding Protein EWS * genetics   MeSH
• Proto-Oncogene Proteins c-myb genetics   MeSH
• Proto-Oncogene Proteins MeSH
• Aged MeSH
• Trans-Activators genetics   MeSH
• NFI Transcription Factors genetics   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

PURPOSE: To assess the intraday repeatability of macular architecture measurements in glaucomatous and non-glaucomatous patients using spectral-domain optical coherence tomography (SD-OCT) and to evaluate the independence from intraindividual intraocular pressure (IOP) fluctuations. METHODS: In this single-center, time-point comparison study, 88 eyes with glaucoma, 53 eyes with ocular hypertension (OHT), and 253 healthy eyes underwent two standardized SD-OCT and intraocular pressure (IOP) measurements on the same day with a 5-h time gap. Bland-Altman plots, intraclass correlation coefficients (ICC), and random-effects model were used to analyze repeatability of entire retinal thickness, retinal nerve fiber layer, ganglion cell layer, inner plexiform layer, and inner nuclear layer measurements. RESULTS: Intraday measurements were highly reproducible in all 3 groups. ICC were greater than 0.90, respectively. The pairwise comparisons of morphometric parameters showed a statistically significant difference (P < 0.001, respectively) between groups (glaucoma vs. control, glaucoma vs. OHT) and a significant influence of time points. No correlation was found between IOP fluctuations and morphometric parameters (P > 0.05, respectively), except for a weak positive correlation with GCL (rho = 0.109, P = 0.031). CONCLUSIONS: The evaluation of macular morphometric parameters of SD-OCT showed a high intraday repeatability and an excellent degree of agreement in glaucoma, ocular hypertension, and healthy groups. The fixed effects of time points were statistically significant. Except for a weak positive correlation of ganglion cell layer, variability did not appear to be affected by intraday IOP changes. Additional research is required to fully understand the impact of IOP fluctuations on macular morphometric parameters, considering the small observed IOP changes.

• MeSH
• Adult MeSH
• Glaucoma * diagnosis   physiopathology   MeSH
• Middle Aged MeSH
• Humans MeSH
• Macula Lutea * pathology   diagnostic imaging   MeSH
• Follow-Up Studies MeSH
• Nerve Fibers * pathology   MeSH
• Intraocular Pressure * physiology   MeSH
• Ocular Hypertension diagnosis   physiopathology   MeSH
• Tomography, Optical Coherence * methods   MeSH
• Reproducibility of Results MeSH
• Retinal Ganglion Cells * pathology   MeSH
• Aged MeSH
• Tonometry, Ocular MeSH
• Visual Fields physiology   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH

ANTXR1 is one of two cell surface receptors mediating the uptake of the anthrax toxin into cells. Despite substantial research on its role in anthrax poisoning and a proposed function as a collagen receptor, ANTXR1's physiological functions remain largely undefined. Pathogenic variants in ANTXR1 lead to the rare GAPO syndrome, named for its four primary features: Growth retardation, Alopecia, Pseudoanodontia, and Optic atrophy. The disease is also associated with a complex range of other phenotypes impacting the cardiovascular, skeletal, pulmonary and nervous systems. Aberrant accumulation of extracellular matrix components and fibrosis are considered to be crucial components in the pathogenesis of GAPO syndrome, contributing to the shortened life expectancy of affected individuals. Nonetheless, the specific mechanisms connecting ANTXR1 deficiency to the clinical manifestations of GAPO syndrome are largely unexplored. In this study, we present evidence that ANTXR1 deficiency initiates a senescent phenotype in human fibroblasts, correlating with defects in nuclear architecture and actin dynamics. We provide novel insights into ANTXR1's physiological functions and propose GAPO syndrome to be reconsidered as a progeroid disorder highlighting an unexpected role for an integrin-like extracellular matrix receptor in human aging.

• MeSH
• Actins metabolism   MeSH
• Alopecia * metabolism   pathology   genetics   MeSH
• Anodontia * MeSH
• Optic Atrophies, Hereditary genetics   metabolism   MeSH
• Fibroblasts * metabolism   MeSH
• Humans MeSH
• Microfilament Proteins * MeSH
• Growth Disorders * MeSH
• Progeria genetics   pathology   metabolism   MeSH
• Receptors, Cell Surface metabolism   genetics   deficiency   MeSH
• Cellular Senescence * genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The unicellular parasite Leishmania has a precisely defined cell architecture that is inherited by each subsequent generation, requiring a highly coordinated pattern of duplication and segregation of organelles and cytoskeletal structures. A framework of nuclear division and morphological changes is known from light microscopy, yet this has limited resolution and the intrinsic organisation of organelles within the cell body and their manner of duplication and inheritance is unknown. Using volume electron microscopy approaches, we have produced three-dimensional reconstructions of different promastigote cell cycle stages to give a spatial and quantitative overview of organelle positioning, division and inheritance. The first morphological indications seen in our dataset that a new cell cycle had begun were the assembly of a new flagellum, the duplication of the contractile vacuole and the increase in volume of the nucleus and kinetoplast. We showed that the progression of the cytokinesis furrow created a specific pattern of membrane indentations, while our analysis of sub-pellicular microtubule organisation indicated that there is likely a preferred site of new microtubule insertion. The daughter cells retained these indentations in their cell body for a period post-abscission. By comparing cultured and sand fly derived promastigotes, we found an increase in the number and overall volume of lipid droplets in the promastigotes from the sand fly, reflecting a change in their metabolism to ensure transmissibility to the mammalian host. Our insights into the cell cycle mechanics of Leishmania will support future molecular cell biology analyses of these parasites.

• MeSH
• Cell Division MeSH
• Cell Cycle MeSH
• Leishmania mexicana * genetics   MeSH
• Leishmania * MeSH
• Parasites * MeSH
• Psychodidae * parasitology   MeSH
• Mammals MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH