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Journal of the American Society of... Feb 2021Membranous nephropathy (MN) occurs due to deposition of immune complexes along the subepithelial region of glomerular basement membrane. Two previously identified target... (Review)
Review
Membranous nephropathy (MN) occurs due to deposition of immune complexes along the subepithelial region of glomerular basement membrane. Two previously identified target antigens for the immune complexes, PLA2R (identified in 2009) and THSD7A (in 2014), account for approximately 60% of all MN, both primary and secondary. In the remaining MN, target antigens were unknown. Use of laser microdissection and mass spectrometry enabled identification of new "antigens." This approach led to the identification of four novel types of MN: exotosin 1 (EXT1)- and exotosin 2 (EXT2)-associated MN, NELL1-associated MN, Sema3B-associated MN, and PCDH7-associated MN. Each of these represents a distinct disease entity, with different clinical and pathologic findings. In this review, the structure of the proteins and the clinical and pathologic findings of the new types of MN are discussed. The role of mass spectrometry for accurate diagnosis of MN cannot be overemphasized. Finally, any classification of MN should be made on the basis of the antigens that are detected. Further studies are required to understand the pathophysiology, response to treatment, and outcomes of these new MNs.
Topics: Cadherins; Calcium-Binding Proteins; Glomerulonephritis, Membranous; Humans; Laser Capture Microdissection; Membrane Glycoproteins; N-Acetylglucosaminyltransferases; Protocadherins; Semaphorins; Tandem Mass Spectrometry
PubMed: 33380523
DOI: 10.1681/ASN.2020071082 -
Nature Biotechnology Aug 2022Despite the availabilty of imaging-based and mass-spectrometry-based methods for spatial proteomics, a key challenge remains connecting images with...
Despite the availabilty of imaging-based and mass-spectrometry-based methods for spatial proteomics, a key challenge remains connecting images with single-cell-resolution protein abundance measurements. Here, we introduce Deep Visual Proteomics (DVP), which combines artificial-intelligence-driven image analysis of cellular phenotypes with automated single-cell or single-nucleus laser microdissection and ultra-high-sensitivity mass spectrometry. DVP links protein abundance to complex cellular or subcellular phenotypes while preserving spatial context. By individually excising nuclei from cell culture, we classified distinct cell states with proteomic profiles defined by known and uncharacterized proteins. In an archived primary melanoma tissue, DVP identified spatially resolved proteome changes as normal melanocytes transition to fully invasive melanoma, revealing pathways that change in a spatial manner as cancer progresses, such as mRNA splicing dysregulation in metastatic vertical growth that coincides with reduced interferon signaling and antigen presentation. The ability of DVP to retain precise spatial proteomic information in the tissue context has implications for the molecular profiling of clinical samples.
Topics: Humans; Laser Capture Microdissection; Mass Spectrometry; Melanoma; Proteome; Proteomics
PubMed: 35590073
DOI: 10.1038/s41587-022-01302-5 -
Nature Neuroscience Aug 2021The human neonatal cerebellum is one-fourth of its adult size yet contains the blueprint required to integrate environmental cues with developing motor, cognitive and...
The human neonatal cerebellum is one-fourth of its adult size yet contains the blueprint required to integrate environmental cues with developing motor, cognitive and emotional skills into adulthood. Although mature cerebellar neuroanatomy is well studied, understanding of its developmental origins is limited. In this study, we systematically mapped the molecular, cellular and spatial composition of human fetal cerebellum by combining laser capture microscopy and SPLiT-seq single-nucleus transcriptomics. We profiled functionally distinct regions and gene expression dynamics within cell types and across development. The resulting cell atlas demonstrates that the molecular organization of the cerebellar anlage recapitulates cytoarchitecturally distinct regions and developmentally transient cell types that are distinct from the mouse cerebellum. By mapping genes dominant for pediatric and adult neurological disorders onto our dataset, we identify relevant cell types underlying disease mechanisms. These data provide a resource for probing the cellular basis of human cerebellar development and disease.
Topics: Cerebellum; Fetus; Humans; Laser Capture Microdissection; Neurogenesis; Single-Cell Analysis; Transcriptome
PubMed: 34140698
DOI: 10.1038/s41593-021-00872-y -
Nature Sep 2021Over the course of an individual's lifetime, normal human cells accumulate mutations. Here we compare the mutational landscape in 29 cell types from the soma and...
Over the course of an individual's lifetime, normal human cells accumulate mutations. Here we compare the mutational landscape in 29 cell types from the soma and germline using multiple samples from the same individuals. Two ubiquitous mutational signatures, SBS1 and SBS5/40, accounted for the majority of acquired mutations in most cell types, but their absolute and relative contributions varied substantially. SBS18, which potentially reflects oxidative damage, and several additional signatures attributed to exogenous and endogenous exposures contributed mutations to subsets of cell types. The rate of mutation was lowest in spermatogonia, the stem cells from which sperm are generated and from which most genetic variation in the human population is thought to originate. This was due to low rates of ubiquitous mutational processes and may be partially attributable to a low rate of cell division in basal spermatogonia. These results highlight similarities and differences in the maintenance of the germline and soma.
Topics: Aged; Clone Cells; Female; Germ Cells; Germ-Line Mutation; Health; Humans; Male; Microdissection; Middle Aged; Mutation Rate; Organ Specificity; Oxidative Stress; Spermatogonia
PubMed: 34433962
DOI: 10.1038/s41586-021-03822-7 -
Nature Methods Oct 2023Single-cell proteomics by mass spectrometry is emerging as a powerful and unbiased method for the characterization of biological heterogeneity. So far, it has been...
Single-cell proteomics by mass spectrometry is emerging as a powerful and unbiased method for the characterization of biological heterogeneity. So far, it has been limited to cultured cells, whereas an expansion of the method to complex tissues would greatly enhance biological insights. Here we describe single-cell Deep Visual Proteomics (scDVP), a technology that integrates high-content imaging, laser microdissection and multiplexed mass spectrometry. scDVP resolves the context-dependent, spatial proteome of murine hepatocytes at a current depth of 1,700 proteins from a cell slice. Half of the proteome was differentially regulated in a spatial manner, with protein levels changing dramatically in proximity to the central vein. We applied machine learning to proteome classes and images, which subsequently inferred the spatial proteome from imaging data alone. scDVP is applicable to healthy and diseased tissues and complements other spatial proteomics and spatial omics technologies.
Topics: Animals; Mice; Proteome; Mass Spectrometry; Proteomics; Laser Capture Microdissection
PubMed: 37783884
DOI: 10.1038/s41592-023-02007-6 -
Journal of Visualized Experiments : JoVE Feb 2021The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain consists of an...
The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain consists of an outer covering of grey matter over the hemispheres known as the cerebral cortex. Underneath this layer reside many other specialized structures that are essential for multiple phenomenon important for existence. Acquiring samples of specific gross brain regions requires quick and precise dissection steps. It is understood that at the microscopic level, many sub-regions exist and likely cross the arbitrary regional boundaries that we impose for the purpose of this dissection. Mouse models are routinely used to study human brain functions and diseases. Changes in gene expression patterns may be confined to specific brain areas targeting a particular phenotype depending on the diseased state. Thus, it is of great importance to study regulation of transcription with respect to its well-defined structural organization. A complete understanding of the brain requires studying distinct brain regions, defining connections, and identifying key differences in the activities of each of these brain regions. A more comprehensive understanding of each of these distinct regions may pave the way for new and improved treatments in the field of neuroscience. Herein, we discuss a step-by-step methodology for dissecting the mouse brain into sixteen distinct regions. In this procedure, we have focused on male mouse C57Bl/6J (6-8 week old) brain removal and dissection into multiple regions using neuroanatomical landmarks to identify and sample discrete functionally-relevant and behaviorally-relevant brain regions. This work will help lay a strong foundation in the field of neuroscience, leading to more focused approaches in the deeper understanding of brain function.
Topics: Animals; Brain; Brain Mapping; Male; Mice, Inbred C57BL; Microdissection; Mice
PubMed: 33645582
DOI: 10.3791/61941 -
Kidney International Jan 2020Membranous nephropathy is characterized by deposition of immune complexes along the glomerular basement membrane. PLA2R and THSD7A are target antigens in 70% and 1-5% of... (Observational Study)
Observational Study
Membranous nephropathy is characterized by deposition of immune complexes along the glomerular basement membrane. PLA2R and THSD7A are target antigens in 70% and 1-5% of primary membranous nephropathy cases, respectively. In the remaining cases, the target antigen is unknown. Here, laser microdissection of glomeruli followed by mass spectrometry was used to identify novel antigen(s) in PLA2R-negative membranous nephropathy. An initial pilot mass spectrometry study in 35 cases of PLA2R-negative membranous nephropathy showed high spectral counts for neural tissue encoding protein with EGF-like repeats, NELL-1, in six cases. Mass spectrometry failed to detect NELL-1 in 23 PLA2R-associated membranous nephropathy and 88 controls. NELL-1 was localized by immunohistochemistry, which showed bright granular glomerular basement membrane staining for NELL-1 in all six cases. Next, an additional 23 NELL-1 positive cases of membranous nephropathy were identified by immunohistochemistry in a discovery cohort of 91 PLA2R-negative membranous nephropathy cases, 14 were confirmed by mass spectrometry. Thus, 29 of 126 PLA2R-negative cases were positive for NELL-1. PLA2R-associated membranous nephropathy and controls stained negative for NELL-1. We then identified five NELL-1 positive cases of membranous nephropathy out of 84 PLA2R and THSD7A-negative cases in two validation cohorts from France and Belgium. By confocal microscopy, both IgG and NELL-1 co-localized to the glomerular basement membrane. Western blot analysis showed reactivity to NELL-1 in five available sera, but no reactivity in control sera. Clinical and biopsy findings of NELL-1 positive membranous nephropathy showed features of primary membranous nephropathy. Thus, a subset of membranous nephropathy is associated with accumulation and co-localization of NELL-1 and IgG along the glomerular basement membrane, and with anti-NELL-1 antibodies in the serum. Hence, NELL-1 defines a distinct type of primary membranous nephropathy.
Topics: Aged; Autoantibodies; Autoantigens; Biopsy; Calcium-Binding Proteins; Case-Control Studies; Cohort Studies; Female; Glomerular Basement Membrane; Glomerulonephritis, Membranous; Humans; Laser Capture Microdissection; Male; Mass Spectrometry; Microscopy, Confocal; Microscopy, Electron; Microscopy, Fluorescence; Middle Aged; Pilot Projects; Receptors, Phospholipase A2; Thrombospondins
PubMed: 31901340
DOI: 10.1016/j.kint.2019.09.014 -
Nature Nov 2022Genome sequencing of cancers often reveals mosaics of different subclones present in the same tumour. Although these are believed to arise according to the principles of...
Genome sequencing of cancers often reveals mosaics of different subclones present in the same tumour. Although these are believed to arise according to the principles of somatic evolution, the exact spatial growth patterns and underlying mechanisms remain elusive. Here, to address this need, we developed a workflow that generates detailed quantitative maps of genetic subclone composition across whole-tumour sections. These provide the basis for studying clonal growth patterns, and the histological characteristics, microanatomy and microenvironmental composition of each clone. The approach rests on whole-genome sequencing, followed by highly multiplexed base-specific in situ sequencing, single-cell resolved transcriptomics and dedicated algorithms to link these layers. Applying the base-specific in situ sequencing workflow to eight tissue sections from two multifocal primary breast cancers revealed intricate subclonal growth patterns that were validated by microdissection. In a case of ductal carcinoma in situ, polyclonal neoplastic expansions occurred at the macroscopic scale but segregated within microanatomical structures. Across the stages of ductal carcinoma in situ, invasive cancer and lymph node metastasis, subclone territories are shown to exhibit distinct transcriptional and histological features and cellular microenvironments. These results provide examples of the benefits afforded by spatial genomics for deciphering the mechanisms underlying cancer evolution and microenvironmental ecology.
Topics: Female; Humans; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Clonal Evolution; Clone Cells; Genomics; Mutation; Tumor Microenvironment; Whole Genome Sequencing; Transcriptome; Reproducibility of Results; Microdissection; Algorithms
PubMed: 36352222
DOI: 10.1038/s41586-022-05425-2 -
Nature Communications Oct 2020Epilepsy is one of the most common neurological disorders, yet its pathophysiology is poorly understood due to the high complexity of affected neuronal circuits. To...
Epilepsy is one of the most common neurological disorders, yet its pathophysiology is poorly understood due to the high complexity of affected neuronal circuits. To identify dysfunctional neuronal subtypes underlying seizure activity in the human brain, we have performed single-nucleus transcriptomics analysis of >110,000 neuronal transcriptomes derived from temporal cortex samples of multiple temporal lobe epilepsy and non-epileptic subjects. We found that the largest transcriptomic changes occur in distinct neuronal subtypes from several families of principal neurons (L5-6_Fezf2 and L2-3_Cux2) and GABAergic interneurons (Sst and Pvalb), whereas other subtypes in the same families were less affected. Furthermore, the subtypes with the largest epilepsy-related transcriptomic changes may belong to the same circuit, since we observed coordinated transcriptomic shifts across these subtypes. Glutamate signaling exhibited one of the strongest dysregulations in epilepsy, highlighted by layer-wise transcriptional changes in multiple glutamate receptor genes and strong upregulation of genes coding for AMPA receptor auxiliary subunits. Overall, our data reveal a neuronal subtype-specific molecular phenotype of epilepsy.
Topics: Adolescent; Adult; Biopsy; Case-Control Studies; Cell Nucleus; Datasets as Topic; Drug Resistant Epilepsy; Epilepsy, Temporal Lobe; Female; Glutamic Acid; Humans; Magnetic Resonance Imaging; Male; Microdissection; Middle Aged; Models, Genetic; Nerve Net; Neurons; RNA-Seq; Receptors, AMPA; Receptors, Glutamate; Signal Transduction; Single-Cell Analysis; Temporal Lobe; Transcription, Genetic; Transcriptome; Up-Regulation; Young Adult
PubMed: 33028830
DOI: 10.1038/s41467-020-18752-7 -
Proteomics May 2021Multiple applications of proteomics in life and health science, pathology and pharmacology, require handling size-limited cell and tissue samples. During proteomic... (Review)
Review
Multiple applications of proteomics in life and health science, pathology and pharmacology, require handling size-limited cell and tissue samples. During proteomic sample preparation, analyte loss in these samples arises when standard procedures are used. Thus, specific considerations have to be taken into account for processing, that are summarised under the term microproteomics (μPs). Microproteomic workflows include: sampling (e.g., flow cytometry, laser capture microdissection), sample preparation (possible disruption of cells or tissue pieces via lysis, protein extraction, digestion in bottom-up approaches, and sample clean-up) and analysis (chromatographic or electrophoretic separation, mass spectrometric measurements and statistical/bioinformatic evaluation). All these steps must be optimised to reach wide protein dynamic ranges and high numbers of identifications. Under optimal conditions, sampling is adapted to the studied sample types and nature, sample preparation isolates and enriches the whole protein content, clean-up removes salts and other interferences such as detergents or chaotropes, and analysis identifies as many analytes as the instrumental throughput and sensitivity allow. In the suggested review, we present and discuss the current state in μP applications for processing of small number of cells (cell μPs) and microscopic tissue regions (tissue μPs).
Topics: Laser Capture Microdissection; Mass Spectrometry; Proteins; Proteomics; Specimen Handling
PubMed: 33547857
DOI: 10.1002/pmic.202000318