High-Throughput Isolation of RNA from Single Cells within Intact Tissue for Spatial and Temporal Sequencing
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE210115 | GEO | 2022/09/30
REPOSITORIES: GEO
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Project description:High-Throughput Isolation of RNA from Single Cells within Intact Tissue for Spatial and Temporal Sequencing
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Project description:The role of human glia in many neurological disorders is still poorly understood due to the lack of tools that reliably isolate specific glial subpopulations from bulk tissue, directly from their native niche. To better understand the contributions of glial pathology in human epilepsy, we developed and validated a novel sorting strategy that simultaneously isolates nuclei populations of astrocytes (PAX6+), oligodendroglial progenitors (OPCs) (OLIG2+) and neurons (NEUN+) from non-pathological fresh-frozen human postmortem temporal neocortex brain tissue (TL Control) and then employed it, in combination with single cell RNA-seq, to characterize the cell-type specific transcriptome alterations in epilepsy temporal neocortex derived from fresh surgical material in patients with medically refractory temporal lobe epilepsy (TLE).
2020-10-29 | GSE140393 | GEO
Project description:Contemporary models of psychosis suggest that a continuum of severity of psychotic symptoms exists, with subthreshold psychotic experiences (PEs) potentially reflecting some genetic and environmental risk factors shared with clinical psychosis. Thus, identifying abnormalities in brain activity that manifest across this continuum can shed new light on the pathophysiology of psychosis. Here, we investigated the moment-to-moment engagement of brain networks ("states") in individuals with schizophrenia (SCZ) and PEs and identified features of these states that are associated with psychosis-spectrum symptoms. Transient brain states were defined by clustering "single snapshots" of blood oxygen level-dependent images, based on spatial similarity of the images. We found that individuals with SCZ (n = 35) demonstrated reduced recruitment of three brain states compared to demographically matched healthy controls (n = 35). Of these three illness-related states, one specific state, involving primarily the visual and salience networks, also occurred at a lower rate in individuals with persistent PEs (n = 22), compared to demographically matched healthy youth (n = 22). Moreover, the occurrence rate of this marker brain state was negatively correlated with the severity of PEs (r = -0.26, p = 0.003, n = 130). In contrast, the spatial map of this state appeared to be unaffected in the SCZ or PE groups. Thus, reduced engagement of a brain state involving the visual and salience networks was demonstrated across the psychosis continuum, suggesting that early disruptions of perceptual and affective function may underlie some of the core symptoms of the illness.
| S-EPMC8286268 | biostudies-literature
Project description:Aims/hypothesisInsulin secretion is widely studied because it plays a central role in glucose homeostasis and diabetes. Processes from insulin granule fusion in beta cells to in vivo insulin secretion have been elucidated, but data at the cellular level do not fully account for several aspects of the macroscopic secretory pattern. Here we investigated how individual secretory events are coordinated spatially and temporally within intact human islets.MethodsWe used the fluorescent probe neuropeptide Y (NPY)-pHluorin to visualise insulin granule secretion in isolated intact human islets.ResultsWe found that individual beta cells respond to increases in glucose concentration by releasing insulin granules in very discrete bursts with periods consistent with in vivo pulsatile insulin secretion. In successive secretory bursts during prolonged exposure to high glucose levels, secretory events progressively localised to preferential release sites, coinciding with the transition to second phase insulin secretion. Granule secretion was very synchronised in neighbouring beta cells, forming discrete regional clusters of activity.Conclusions/interpretationThese results reveal how individual secretory events are coordinated to produce pulsatile insulin secretion from human islets.
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Project description:Highly localized (point) constrictions featuring a round geometry with ultra-sharp edges in silicon have been demonstrated for the reagent-free continuous-flow rapid mechanical lysis of mammalian cells on a single-cell basis. Silicon point constrictions, robust structures formed by a single-step dry etching process, are arranged in a cascade along microfluidic channels and can effectively rupture cells delivered in a pressure-driven flow. The influence of the constriction size and count on the lysis performance is presented for fibroblasts in reference to total protein, DNA, and intact nuclei levels in the lysates evaluated by biochemical and fluoremetric assays and flow-cytometric analyses. Protein and DNA levels obtained from an eight-constriction treatment match or surpass those from a chemical method. More importantly, many intact nuclei are found in the lysates with a relatively high nuclei-isolation efficiency from a four-constriction treatment. Point constrictions and their role in rapid reagent-free disruption of the plasma membrane could have implications for integrated sample preparation in future lab-on-a-chip systems.
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Project description:Hydrogen peroxide (H2O2) has key signaling roles at physiological levels, while causing molecular damage at elevated concentrations. H2O2 production by mitochondria is implicated in regulating processes inside and outside these organelles. However, it remains unclear whether and how mitochondria in intact cells release H2O2. Here, we employed a genetically-encoded high-affinity H2O2 sensor, HyPer7, in mammalian tissue culture cells to investigate different modes of mitochondrial H2O2 release. We found substantial heterogeneity of HyPer7 dynamics between individual cells. We further observed mitochondria-released H2O2 directly at the surface of the organelle and in the bulk cytosol, but not in the nucleus or at the plasma membrane, pointing to steep gradients emanating from mitochondria. Gradient formation is controlled by cytosolic peroxiredoxins, which act redundantly and with a substantial reserve capacity. Dynamic adaptation of cytosolic thioredoxin reductase levels during metabolic changes results in improved H2O2 handling and explains previously-observed differences between cell types. Our data suggest that H2O2-mediated signaling is initiated only in close proximity to mitochondria and under specific metabolic conditions.
| S-SCDT-EMBOJ-2021-109169 | biostudies-other
Project description:High space-bandwidth product with high spatial phase sensitivity is indispensable for a single-shot quantitative phase microscopy (QPM) system. It opens avenue for widespread applications of QPM in the field of biomedical imaging. Temporally low coherence light sources are implemented to achieve high spatial phase sensitivity in QPM at the cost of either reduced temporal resolution or smaller field of view (FOV). In addition, such light sources have low photon degeneracy. On the contrary, high temporal coherence light sources like lasers are capable of exploiting the full FOV of the QPM systems at the expense of less spatial phase sensitivity. In the present work, we demonstrated that use of narrowband partially spatially coherent light source also called pseudo-thermal light source (PTLS) in QPM overcomes the limitations of conventional light sources. The performance of PTLS is compared with conventional light sources in terms of space bandwidth product, phase sensitivity and optical imaging quality. The capabilities of PTLS are demonstrated on both amplitude (USAF resolution chart) and phase (thin optical waveguide, height ~ 8 nm) objects. The spatial phase sensitivity of QPM using PTLS is measured to be equivalent to that for white light source and supports the FOV (18 times more) equivalent to that of laser light source. The high-speed capabilities of PTLS based QPM is demonstrated by imaging live sperm cells that is limited by the camera speed and large FOV is demonstrated by imaging histopathology human placenta tissue samples. Minimal invasive, high-throughput, spatially sensitive and single-shot QPM based on PTLS will enable wider penetration of QPM in life sciences and clinical applications.
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Project description:We established a high-depth spatial transcriptomics technology, photo-isolation chemistry (PIC; Honda, et al., Nat Commun, 2021), which is able to isolate gene expression profiles only from photo-irradiated region out of whole tissues. In the original protocol, only the fresh-frozen sections were available, but we have now optimized it applicable to formalin-fixed frozen and formalin-fixed paraffin sections. Here, we provide an updated protocol of PIC.
2022-05-04 | GSE195459 | GEO
Project description:BackgroundParaHox and Hox genes are thought to have evolved from a common ancestral ProtoHox cluster or from tandem duplication prior to the divergence of cnidarians and bilaterians. Similar to Hox clusters, chordate ParaHox genes including Gsx, Xlox, and Cdx, are clustered and their expression exhibits temporal and spatial colinearity. In non-chordate animals, however, studies on the genomic organization of ParaHox genes are limited to only a few animal taxa. Hemichordates, such as the Enteropneust acorn worms, have been used to gain insights into the origins of chordate characters. In this study, we investigated the genomic organization and expression of ParaHox genes in the indirect developing hemichordate acorn worm Ptychodera flava.ResultsWe found that P. flava contains an intact ParaHox cluster with a similar arrangement to that of chordates. The temporal expression order of the P. flava ParaHox genes is the same as that of the chordate ParaHox genes. During embryogenesis, the spatial expression pattern of PfCdx in the posterior endoderm represents a conserved feature similar to the expression of its orthologs in other animals. On the other hand, PfXlox and PfGsx show a novel expression pattern in the blastopore. Nevertheless, during metamorphosis, PfXlox and PfCdx are expressed in the endoderm in a spatially staggered pattern similar to the situation in chordates.ConclusionsOur study shows that P. flava ParaHox genes, despite forming an intact cluster, exhibit temporal colinearity but lose spatial colinearity during embryogenesis. During metamorphosis, partial spatial colinearity is retained in the transforming larva. These results strongly suggest that intact ParaHox gene clustering was retained in the deuterostome ancestor and is correlated with temporal colinearity.
| S-EPMC3698058 | biostudies-literature
Project description:Current dissociation methods for scRNA-seq studies of solid tissues do not guarantee intact single-cell isolation from fresh frozen samples, especially for sensitive and heterogeneous endocrine tissues. Here, we adapted the acetic-methanol dissociation method – ACME High Soult (ACME HS) to isolate intact single cells from fresh-frozen endocrine tumor samples. We compared enzymatic, ACME HS, and nuclear isolation methods ability to preserve major cell types and gene expression. We demonstrated that ACME HS dissociates and fixes cells, preserving cell morphology and high RNA integrity. This renders ACME HS a valuable alternative in the scRNA-seq protocols for challenging tissues.
2025-01-08 | GSE264080 | GEO