A petavoxel fragment of human cerebral cortex reconstructed at nanoscale resolution.

To fully understand how the human brain works, knowledge of its structure at high resolution is needed. Presented here is a computationally intensive reconstruction of the ultrastructure of a cubic millimeter of human temporal cortex that was surgically removed to gain access to an underlying epileptic focus. It contains about 57,000 cells, about 230 millimeters of blood vessels, and about 150 million synapses and comprises 1.4 petabytes. Our analysis showed that glia outnumber neurons 2:1, oligodendrocytes were the most common cell, deep layer excitatory neurons could be classified on the basis of dendritic orientation, and among thousands of weak connections to each neuron, there exist rare powerful axonal inputs of up to 50 synapses. Further studies using this resource may bring valuable insights into the mysteries of the human brain.

Preserving extracellular space for high-quality optical and ultrastructural studies of whole mammalian brains.

Analysis of brain structure, connectivity, and molecular diversity relies on effective tissue fixation. Conventional tissue fixation causes extracellular space (ECS) loss, complicating the segmentation of cellular objects from electron microscopy datasets. Previous techniques for preserving ECS in mammalian brains utilizing high-pressure perfusion can give inconsistent results owing to variations in the hydrostatic pressure within the vasculature. A more reliable fixation protocol that uniformly preserves the ECS throughout whole brains would greatly benefit a wide range of neuroscience studies. Here, we report a straightforward transcardial perfusion strategy that preserves ECS throughout the whole rodent brain. No special setup is needed besides sequential solution changes, and the protocol offers excellent reproducibility. In addition to better capturing tissue ultrastructure, preservation of ECS has many downstream advantages such as accelerating heavy-metal staining for electron microscopy, improving detergent-free immunohistochemistry for correlated light and electron microscopy, and facilitating lipid removal for tissue clearing.

TGFBR3L is associated with gonadotropin production in non-functioning gonadotroph pituitary neuroendocrine tumours.

PURPOSE: Transforming growth factor-beta receptor 3-like (TGFBR3L) is a pituitary enriched membrane protein selectively detected in gonadotroph cells. TGFBR3L is named after transforming growth factor-beta receptor 3 (TGFBR3), an inhibin A co-receptor in mice, due to sequence identity to the C-terminal region. We aimed to characterize TGFBR3L detection in a well-characterized, prospectively collected cohort of non-functioning pituitary neuroendocrine tumours (NF-PitNETs) and correlate it to clinical data. METHODS: 144 patients operated for clinically NF-PitNETs were included. Clinical, radiological and biochemical data were recorded. Immunohistochemical (IHC) staining for FSHß and LHß was scored using the immunoreactive score (IRS), TGFBR3L and TGFBR3 were scored by the percentage of positive stained cells. RESULTS: TGFBR3L staining was selectively present in 52% of gonadotroph tumours. TGFBR3L was associated to IRS of LHß (median 2 [IQR 0-3] in TGFBR3L negative and median 6 [IQR 3-9] in TGFBR3L positive tumours, p < 0.001), but not to the IRS of FSHß (p = 0.32). The presence of TGFBR3L was negatively associated with plasma gonadotropin concentrations in males (P-FSH median 5.5 IU/L [IQR 2.9-9.6] and median 3.0 [IQR 1.8-5.6] in TGFBR3L negative and positive tumours respectively, p = 0.008) and P-LH (median 2.8 IU/L [IQR 1.9-3.7] and median 1.8 [IQR 1.1-3.0] in TGFBR3L negative and positive tumours respectively, p = 0.03). TGFBR3 stained positive in 22% (n = 25) of gonadotroph tumours with no correlation to TGFBR3L. CONCLUSION: TGFBR3L was selectively detected in half (52%) of gonadotroph NF-PitNETs. The association to LHß staining and plasma gonadotropins suggests that TGFBR3L may be involved in hormone production in gonadotroph NF-PitNETs.

Endothelial cell heterogeneity and microglia regulons revealed by a pig cell landscape at single-cell level.

Pigs are valuable large animal models for biomedical and genetic research, but insights into the tissue- and cell-type-specific transcriptome and heterogeneity remain limited. By leveraging single-cell RNA sequencing, we generate a multiple-organ single-cell transcriptomic map containing over 200,000 pig cells from 20 tissues/organs. We comprehensively characterize the heterogeneity of cells in tissues and identify 234 cell clusters, representing 58 major cell types. In-depth integrative analysis of endothelial cells reveals a high degree of heterogeneity. We identify several functionally distinct endothelial cell phenotypes, including an endothelial to mesenchymal transition subtype in adipose tissues. Intercellular communication analysis predicts tissue- and cell type-specific crosstalk between endothelial cells and other cell types through the VEGF, PDGF, TGF-ß, and BMP pathways. Regulon analysis of single-cell transcriptome of microglia in pig and 12 other species further identifies MEF2C as an evolutionally conserved regulon in the microglia. Our work describes the landscape of single-cell transcriptomes within diverse pig organs and identifies the heterogeneity of endothelial cells and evolutionally conserved regulon in microglia.

Genome-wide annotation of protein-coding genes in pig.

BACKGROUND: There is a need for functional genome-wide annotation of the protein-coding genes to get a deeper understanding of mammalian biology. Here, a new annotation strategy is introduced based on dimensionality reduction and density-based clustering of whole-body co-expression patterns. This strategy has been used to explore the gene expression landscape in pig, and we present a whole-body map of all protein-coding genes in all major pig tissues and organs. RESULTS: An open-access pig expression map ( www.rnaatlas.org ) is presented based on the expression of 350 samples across 98 well-defined pig tissues divided into 44 tissue groups. A new UMAP-based classification scheme is introduced, in which all protein-coding genes are stratified into tissue expression clusters based on body-wide expression profiles. The distribution and tissue specificity of all 22,342 protein-coding pig genes are presented. CONCLUSIONS: Here, we present a new genome-wide annotation strategy based on dimensionality reduction and density-based clustering. A genome-wide resource of the transcriptome map across all major tissues and organs in pig is presented, and the data is available as an open-access resource ( www.rnaatlas.org ), including a comparison to the expression of human orthologs.

A single-cell type transcriptomics map of human tissues.

Advances in molecular profiling have opened up the possibility to map the expression of genes in cells, tissues, and organs in the human body. Here, we combined single-cell transcriptomics analysis with spatial antibody-based protein profiling to create a high-resolution single-cell type map of human tissues. An open access atlas has been launched to allow researchers to explore the expression of human protein-coding genes in 192 individual cell type clusters. An expression specificity classification was performed to determine the number of genes elevated in each cell type, allowing comparisons with bulk transcriptomics data. The analysis highlights distinct expression clusters corresponding to cell types sharing similar functions, both within the same organs and between organs.

A porcine brain-wide RNA editing landscape.

Adenosine-to-inosine (A-to-I) RNA editing, catalyzed by ADAR enzymes, is an essential post-transcriptional modification. Although hundreds of thousands of RNA editing sites have been reported in mammals, brain-wide analysis of the RNA editing in the mammalian brain remains rare. Here, a genome-wide RNA-editing investigation is performed in 119 samples, representing 30 anatomically defined subregions in the pig brain. We identify a total of 682,037 A-to-I RNA editing sites of which 97% are not identified before. Within the pig brain, cerebellum and olfactory bulb are regions with most edited transcripts. The editing level of sites residing in protein-coding regions are similar across brain regions, whereas region-distinct editing is observed in repetitive sequences. Highly edited conserved recoding events in pig and human brain are found in neurotransmitter receptors, demonstrating the evolutionary importance of RNA editing in neurotransmission functions. Although potential data biases caused by age, sex or health status are not considered, this study provides a rich resource to better understand the evolutionary importance of post-transcriptional RNA editing.

Association of CSF proteins with tau and amyloid ß levels in asymptomatic 70-year-olds.

BACKGROUND: Increased knowledge of the evolution of molecular changes in neurodegenerative disorders such as Alzheimer's disease (AD) is important for the understanding of disease pathophysiology and also crucial to be able to identify and validate disease biomarkers. While several biological changes that occur early in the disease development have already been recognized, the need for further characterization of the pathophysiological mechanisms behind AD still remains. METHODS: In this study, we investigated cerebrospinal fluid (CSF) levels of 104 proteins in 307 asymptomatic 70-year-olds from the H70 Gothenburg Birth Cohort Studies using a multiplexed antibody- and bead-based technology. RESULTS: The protein levels were first correlated with the core AD CSF biomarker concentrations of total tau, phospho-tau and amyloid beta (Aß42) in all individuals. Sixty-three proteins showed significant correlations to either total tau, phospho-tau or Aß42. Thereafter, individuals were divided based on CSF Aß42/Aß40 ratio and Clinical Dementia Rating (CDR) score to determine if early changes in pathology and cognition had an effect on the correlations. We compared the associations of the analysed proteins with CSF markers between groups and found 33 proteins displaying significantly different associations for amyloid-positive individuals and amyloid-negative individuals, as defined by the CSF Aß42/Aß40 ratio. No differences in the associations could be seen for individuals divided by CDR score. CONCLUSIONS: We identified a series of transmembrane proteins, proteins associated with or anchored to the plasma membrane, and proteins involved in or connected to synaptic vesicle transport to be associated with CSF biomarkers of amyloid and tau pathology in AD. Further studies are needed to explore these proteins' role in AD pathophysiology.

Cardiovascular consequences of parathyroid disorders in adults.

PTH is a metabolic active hormone primarily regulating calcium and phosphate homeostasis in a very tight and short term-manner. Parathyroid disorders in adult patients reflect a variety of different conditions related either to the parathyroid glands itself or to the effects of the secreted hormone. The clinical spectrum varies from the common disease primary hyperparathyroidism (PHPT) to the orphan conditions pseudohypoparathyroidism (Ps-HypoPT) and chronic hypoparathyroidism (HypoPT). The purpose of this review is to describe the consequences of disturbances in levels or action of PTH for cardiac function and cardiovascular risk in adult patients with these disorders. Most patients with PHPT achieve the diagnose by chance and have minor or no specific symptoms. Still, these patients with mild PHPT do possess cardiovascular (CV) morbidity, however so far not proven ameliorated by surgery in controlled trials. In severe cases, the CV risk is increased and with a potential reversibility by treatment. Patients with Ps-HypoPT have resistance to PTH action, but not necessarily total resistance in all tissues. So far, no clear CV morbidity or risk has been demonstrated, but there are several aspects of interest for further studies. Most patients with HypoPT do get their hormonal deficiency syndrome following neck surgery. These patients do experience multiple symptoms and do have an increased CV-risk before the primary surgery. Based on existing data, their CV mortality do not deviate from the expected when adjusting for the preexisting increased risk. Patients with nonsurgical (NS-) HypoPT do demonstrate increased CV-risk also associated with exposure time. Endocrine disorders with alterations in PTH function have major impact on the cardiovascular system of importance for morbidity and mortality, wherefore management of these specific diseases should be optimized currently, as new data become available, however also avoiding over-treating asymptomatic patients.

Enhanced Validation of Antibodies Enables the Discovery of Missing Proteins.

The localization of proteins at a tissue- or cell-type-specific level is tightly linked to the protein function. To better understand each protein's role in cellular systems, spatial information constitutes an important complement to quantitative data. The standard methods for determining the spatial distribution of proteins in single cells of complex tissue samples make use of antibodies. For a stringent analysis of the human proteome, we used orthogonal methods and independent antibodies to validate 5981 antibodies that show the expression of 3775 human proteins across all major human tissues. This enhanced validation uncovered 56 proteins corresponding to the group of "missing proteins" and 171 proteins of unknown function. The presented strategy will facilitate further discussions around criteria for evidence of protein existence based on immunohistochemistry and serves as a useful guide to identify candidate proteins for integrative studies with quantitative proteomics methods.

An atlas of the protein-coding genes in the human, pig, and mouse brain.

The brain, with its diverse physiology and intricate cellular organization, is the most complex organ of the mammalian body. To expand our basic understanding of the neurobiology of the brain and its diseases, we performed a comprehensive molecular dissection of 10 major brain regions and multiple subregions using a variety of transcriptomics methods and antibody-based mapping. This analysis was carried out in the human, pig, and mouse brain to allow the identification of regional expression profiles, as well as to study similarities and differences in expression levels between the three species. The resulting data have been made available in an open-access Brain Atlas resource, part of the Human Protein Atlas, to allow exploration and comparison of the expression of individual protein-coding genes in various parts of the mammalian brain.

TGFBR3L-An Uncharacterised Pituitary Specific Membrane Protein Detected in the Gonadotroph Cells in Non-Neoplastic and Tumour Tissue.

Here, we report the investigation of transforming growth factor beta-receptor 3 like (TGFBR3L), an uncharacterised pituitary specific membrane protein, in non-neoplastic anterior pituitary gland and pituitary neuroendocrine tumours. A polyclonal antibody produced within the Human Protein Atlas project (HPA074356) was used for TGFBR3L staining and combined with SF1 and FSH for a 3-plex fluorescent protocol, providing more details about the cell lineage specificity of TGFBR3L expression. A cohort of 230 pituitary neuroendocrine tumours were analysed. In a subgroup of previously characterised gonadotroph tumours, correlation with expression of FSH/LH, E-cadherin, oestrogen (ER) and somatostatin receptors (SSTR) was explored. TGFBR3L showed membranous immunolabeling and was found to be gonadotroph cell lineage-specific, verified by co-expression with SF1 and FSH/LH staining in both tumour and non-neoplastic anterior pituitary tissues. TGFBR3L immunoreactivity was observed in gonadotroph tumours only and demonstrated intra-tumour heterogeneity with a perivascular location. TGFBR3L immunostaining correlated positively to both FSH (R = 0.290) and LH (R = 0.390) immunostaining, and SSTR3 (R = 0.315). TGFBR3L correlated inversely to membranous E-cadherin staining (R = -0.351) and oestrogen receptor ß mRNA (R = -0.274). In conclusion, TGFBR3L is a novel pituitary gland specific protein, located in the membrane of gonadotroph cells in non-neoplastic anterior pituitary gland and in a subset of gonadotroph pituitary tumours.

A genome-wide transcriptomic analysis of protein-coding genes in human blood cells.

Blood is the predominant source for molecular analyses in humans, both in clinical and research settings. It is the target for many therapeutic strategies, emphasizing the need for comprehensive molecular maps of the cells constituting human blood. In this study, we performed a genome-wide transcriptomic analysis of protein-coding genes in sorted blood immune cell populations to characterize the expression levels of each individual gene across the blood cell types. All data are presented in an interactive, open-access Blood Atlas as part of the Human Protein Atlas and are integrated with expression profiles across all major tissues to provide spatial classification of all protein-coding genes. This allows for a genome-wide exploration of the expression profiles across human immune cell populations and all major human tissues and organs.

The human secretome.

The proteins secreted by human cells (collectively referred to as the secretome) are important not only for the basic understanding of human biology but also for the identification of potential targets for future diagnostics and therapies. Here, we present a comprehensive analysis of proteins predicted to be secreted in human cells, which provides information about their final localization in the human body, including the proteins actively secreted to peripheral blood. The analysis suggests that a large number of the proteins of the secretome are not secreted out of the cell, but instead are retained intracellularly, whereas another large group of proteins were identified that are predicted to be retained locally at the tissue of expression and not secreted into the blood. Proteins detected in the human blood by mass spectrometry-based proteomics and antibody-based immunoassays are also presented with estimates of their concentrations in the blood. The results are presented in an updated version 19 of the Human Protein Atlas in which each gene encoding a secretome protein is annotated to provide an open-access knowledge resource of the human secretome, including body-wide expression data, spatial localization data down to the single-cell and subcellular levels, and data about the presence of proteins that are detectable in the blood.

Integration of Transcriptomics and Antibody-Based Proteomics for Exploration of Proteins Expressed in Specialized Tissues.

A large portion of human proteins are referred to as missing proteins, defined as protein-coding genes that lack experimental data on the protein level due to factors such as temporal expression, expression in tissues that are difficult to sample, or they actually do not encode functional proteins. In the present investigation, an integrated omics approach was used for identification and exploration of missing proteins. Transcriptomics data from three different sources-the Human Protein Atlas (HPA), the GTEx consortium, and the FANTOM5 consortium-were used as a starting point to identify genes selectively expressed in specialized tissues. Complementing the analysis with profiling on more specific tissues based on immunohistochemistry allowed for further exploration of cell-type-specific expression patterns. More detailed tissue profiling was performed for >300 genes on complementing tissues. The analysis identified tissue-specific expression of nine proteins previously listed as missing proteins (POU4F1, FRMD1, ARHGEF33, GABRG1, KRTAP2-1, BHLHE22, SPRR4, AVPR1B, and DCLK3), as well as numerous proteins with evidence of existence on the protein level that previously lacked information on spatial resolution and cell-type-specific expression pattern. We here present a comprehensive strategy for identification of missing proteins by combining transcriptomics with antibody-based proteomics. The analyzed proteins provide interesting targets for organ-specific research in health and disease.

A pathology atlas of the human cancer transcriptome.

Cancer is one of the leading causes of death, and there is great interest in understanding the underlying molecular mechanisms involved in the pathogenesis and progression of individual tumors. We used systems-level approaches to analyze the genome-wide transcriptome of the protein-coding genes of 17 major cancer types with respect to clinical outcome. A general pattern emerged: Shorter patient survival was associated with up-regulation of genes involved in cell growth and with down-regulation of genes involved in cellular differentiation. Using genome-scale metabolic models, we show that cancer patients have widespread metabolic heterogeneity, highlighting the need for precise and personalized medicine for cancer treatment. All data are presented in an interactive open-access database (www.proteinatlas.org/pathology) to allow genome-wide exploration of the impact of individual proteins on clinical outcomes.

A subcellular map of the human proteome.

Resolving the spatial distribution of the human proteome at a subcellular level can greatly increase our understanding of human biology and disease. Here we present a comprehensive image-based map of subcellular protein distribution, the Cell Atlas, built by integrating transcriptomics and antibody-based immunofluorescence microscopy with validation by mass spectrometry. Mapping the in situ localization of 12,003 human proteins at a single-cell level to 30 subcellular structures enabled the definition of the proteomes of 13 major organelles. Exploration of the proteomes revealed single-cell variations in abundance or spatial distribution and localization of about half of the proteins to multiple compartments. This subcellular map can be used to refine existing protein-protein interaction networks and provides an important resource to deconvolute the highly complex architecture of the human cell.

Mass spectrometric analysis of synaptosomal membrane preparations for the determination of brain receptors, transporters and channels.

The molecular composition of synaptic signal transduction machineries shapes synaptic neurotransmission. The repertoire of receptors, transporters and channels (RTCs) comprises major signaling events in the brain. RTCs are conventionally studied by candidate immunohistochemistry and biochemistry, which are low throughput with resolution greatly affected by available immunoreagents and membrane interference. Therefore, a comprehensive resource of synaptic brain RTCs is still lacking. In particular, studies on the detergent-soluble synaptosomal fraction, known to contain transporters and channels, are limited. We, therefore, performed sub-synaptosomal fractionation of rat cerebral cortex, followed by trypsin/chymotrypsin sequential digestion of a detergent-soluble synaptosomal fraction and a postsynaptic density preparation, stable-isotope tryptic peptide labeling and liquid chromatography mass spectrometry. Based on the current study, a total of 4784 synaptic proteins were submitted to the ProteomExchange database (PXD001948), including 274 receptors, 394 transporters/channels and 1377 transmembrane proteins. Function-based classification assigned 1781 proteins as probable drug targets with 834 directly linked to brain disorders. The analytical approach identified 499 RTCs that are not listed in the largest, curated database for synaptosomal proteins (SynProt). This is a threefold RTC increase over all other data collected to date. Taken together, we present a protein discovery resource that can serve as a benchmark for future molecular interrogation of synaptic connectivity.