Single-cell Deletion Research Articles

Single Cell Deletion of the Transcription Factors Trps1 and Sox9 in Astrocytes Reveals Novel Functions in the Adult Cerebral Cortex.

Astrocytes play key roles in brain function, but how these are orchestrated by transcription factors (TFs) in the adult brain and aligned with astrocyte heterogeneity is largely unknown. Here we examined the localization and function of the novel astrocyte TF Trps1 (Transcriptional Repressor GATA Binding 1) and the well-known astrocyte TF Sox9 by Cas9-mediated deletion using Mokola-pseudotyped lentiviral delivery into the adult cerebral cortex. Trps1 and Sox9 levels showed heterogeneity among adult cortical astrocytes, which prompted us to explore the effects of deleting either Sox9 or Trps1 alone or simultaneously at the single-cell (by patch-based single-cell transcriptomics) and tissue levels (by spatial transcriptomics). This revealed TF-specific functions in astrocytes, such as synapse maintenance with the strongest effects on synapse number achieved by Trps1 deletion and a common effect on immune response. In addition, spatial transcriptomics showed non-cell-autonomous effects on the surrounding cells, such as oligodendrocytes and other immune cells with TF-specific differences on the type of immune cells: Trps1 deletion affecting monocytes specifically, while Sox9 deletion acting mostly on microglia and deletion of both TF affecting mostly B cells. Taken together, this study reveals novel roles of Trps1 and Sox9 in adult astrocytes and their communication with other glial and immune cells.

Optimizing fault tolerance of RAM cell through MUX based modeling and design using symmetries of QCA cells

Extensive research is now being conducted on the design and construction of logic circuits utilizing quantum-dot cellular automata (QCA) technology. This area of study is of great interest due to the inherent advantages it offers, such as its compact size, high speed, low power dissipation, and enhanced switching frequency in the nanoscale domain. This work presents a design of a highly efficient RAM cell in QCA, utilizing a combination of a 3-input and 5-input Majority Voter (MV) gate, together with a 2 × 1 Multiplexer (MUX). The proposed design is also investigated for various faults such as single cell deletion, single cell addition and single cell displacement or misalignment defects. The circuit under consideration has a high degree of fault tolerance. The functionality of the suggested design is showcased and verified through the utilization of the QCADesigner tool. Based on the observed performance correlation, it is evident that the proposed design demonstrates effectiveness in terms of cell count, area, and latency. Furthermore, it achieves a notable improvement of up to 76.72% compared to the present configuration in terms of quantum cost. The analysis of energy dissipation, conducted using the QCAPro tool, is also shown for various scenarios. It is seen that this design exhibits the lowest energy dispersion, hence enabling the development of ultra-low power designs for diverse microprocessors and microcontrollers.

Cleavage Blastomere Deletion and Transplantation to Test Cell Fate Commitment in Xenopus.

Fate maps identify the precursors of an organ, and tracing the members of a blastomere lineage over time shows how its descendants come to populate that organ. The fates of the individual blastomeres of the two- to 32-cell Xenopus embryo have been fully mapped to reveal which cells are the major contributors to various cell types, tissues, and organs. However, because these fate maps were produced in the normal embryo, they do not reveal whether a precursor blastomere is competent to give rise to additional tissues or is already committed to its fate-mapped repertoire of descendants. To identify the mechanisms by which a cell's fate is committed, one needs to expose the cell to different experimental environments. If the cell's fate is determined, it will express its normal fate or gene expression profile in novel environments, whereas if it is not yet determined it will express different fates or gene expression profiles when exposed to novel external factors or neighboring cells. This protocol describes two techniques for testing cell fate commitment: single cell deletion and single cell transplantation. Deleting a blastomere allows one to test whether the deleted cell is required for the remaining cells to produce their normal, specific cell fates. Transplanting a blastomere to a novel location in a host embryo allows one to test whether the transplanted cell is committed to produce its normal fate-mapped repertoire, or whether it is still competent to respond to novel cell-cell interactions.

Postsynaptic mGluR5 promotes evoked AMPAR-mediated synaptic transmission onto neocortical layer 2/3 pyramidal neurons during development.

Both short- and long-term roles for the group I metabotropic glutamate receptor number 5 (mGluR5) have been examined for the regulation of cortical glutamatergic synapses. However, how mGluR5 sculpts neocortical networks during development still remains unclear. Using a single cell deletion strategy, we examined how mGluR5 regulates glutamatergic synaptic pathways in neocortical layer 2/3 (L2/3) during development. Electrophysiological measurements were made in acutely prepared slices to obtain a functional understanding of the effects stemming from loss of mGluR5 in vivo. Loss of postsynaptic mGluR5 results in an increase in the frequency of action potential-independent synaptic events but, paradoxically, results in a decrease in evoked transmission in two separate synaptic pathways providing input to the same pyramidal neurons. Synaptic transmission through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, but not N-methyl-d-aspartate (NMDA) receptors, is specifically decreased. In the local L2/3 pathway, the decrease in evoked transmission appears to be largely due to a decrease in cell-to-cell connectivity and not in the strength of individual cell-to-cell connections. This decrease in evoked transmission correlates with a decrease in the total dendritic length in a region of the dendritic arbor that likely receives substantial input from these two pathways, thereby suggesting a morphological correlate to functional alterations. These changes are accompanied by an increase in intrinsic membrane excitability. Our data indicate that total mGluR5 function, incorporating both short- and long-term processes, promotes the strengthening of AMPA receptor-mediated transmission in multiple neocortical pathways.

The Role of Apoptosis in Gynaecological Malignancies

Apoptosis is a process of single-cell deletion requiring active participation of the cell in its own demise. First described in 1972, it is now known to play a major role in embryogenesis, tissue homeostasis and neoplasia. Apoptosis can be initiated when DNA damage occurs causing the cell to pause in its reproductive cycle. If the DNA damage is beyond repair, the cell proceeds to apoptotic cell death. When the genetic mechanism(s) involved in the pathway of apoptosis is altered, the cell does not die. Further mutations occur by proliferation and such multiple mutational events can lead to a malignant phenotype and cancer growth. The tumour suppressor gene p53 causes a DNA-damaged cell to rest and attempt repair. If damage is irreparable, p53 levels will continue to increase, initiating apoptosis. Mutation of p53, found in approximately 50% of cancers, can stop the apoptotic process. Increased bcl-2 expression, an apoptosis inhibitor, also plays a role in cellular transformation and cancer growth. Its altered expression occurs in the presence of oncogene expression. This paper reviews the role of apoptosis in malignant transformation, cancer growth, and response to therapy for gynaecological cancers. For cervical cancer and its precursors, data on apoptotic index, bcl-2 and Bax expression are presented and discussed in relationship to human papillomavirus expression. In ovarian epithelial malignancies, the role that apoptosis plays in chemotherapeutic responses is reviewed. The data for endometrial cancer are currently limited to apoptotic index.

Simple developmental programs of gene expression and cellular composition of lymphoid organs at the origin of natural tolerance

Self-tolerance is acquired in the embryonic/perinatal period, but new lymphocytes (that will have to distinguish between self and nonself) continue to be produced throughout life, after both self and nonself are present. This makes it impossible for natural tolerance to rely on recessive mechanisms. Recent observations on "dominant tolerance" have led to the hypothesis that natural tolerance is established as a consequence of simple developmental programs for gene expression and cellular composition of primary lymphoid organs. In development, the cellular composition of the thymus is predominantly epithelial, allowing for the positive selection and activation of "high avidity" self-reactive T cells that are not deleted because antigen presentation by haemopoietic cells is limiting. Such T cells, activated in that environment, display effector functions of a regulatory type that are maintained in the periphery upon restimulation by tissue peptides shared with the thymic epithelium. Recent thymic emigrants with specificity for tissue-specific antigens that are absent from the thymus will first encounter their ligands in the context of the "regulatory cell" recognition of ubiquitous peptides and are thus recruited into similar regulatory activities. In contrast, thymic emigrants with specificity for nonself antigens (absent during the perinatal period) are not activated intrathymically, reach the periphery as resting cells, and move out of the time window of susceptibility to functional recruitment. These will react "de novo" upon encounter of the respective antigens and will acquire the class of effector functions determined by the peripheral microenvironment in which they are activated. This strategy, which explains the thymic dependence of peripheral tissue-specific tolerance, may be re-enforced by developmental restrictions in cytokine gene expression, and it will ensure the establishment and maintenance of T-cell tolerance through the dynamic storage of a distributed memory of the embryonic self. B lymphocytes that are produced in the embryonic/perinatal period characteristically rearrange and express a few V-genes very predominantly. These V-genes encode antibodies with unique properties of "connectivity" to other V-regions, making it possible to establish a network that limits clonal expansions and/or terminal differentiation to antibody production. Self-reactive B cells are thus recruited into such a network which, by contributing to the molecular environment of the body and to the selection of emergent repertoires, leads to deletion of connected cells and to the "normalization" of the adult antibody repertoires. Natural autoantibody repertoires in the adult are thus recursively maintained, stable and continuously adjusted to the thresholds of single cell deletion and to the alterations in the body composition. The activity of self-specific regulatory T cells contributes to limiting clonal expansion and inhibiting somatic mutation of self-reactive B cells. This model explains a number of observations that were not included in the "clonal" and "recessive" tolerance views, and offers suggestions on mechanisms in physiological autoimmunity and pathology.

Kinetic studies on a murine sarcoma and an analysis of apoptosis.

A stathmokinetic method has been used to determine the cell cycle parameters, particularly the potential tumour doubling time, of a murine fat pad sarcoma. Additional information has been obtained by determining the percentage of labelled mitoses (PLM). A technique which simultaneously demonstrates autoradiographically labelled S phase nuclei and histochemically localized acid phosphatase activity has also been used at light microscope level to compare these parameters: acid phosphatase activity was demonstrated in tumour cells and macrophages. Single cell deletion by apoptosis has been investigated as distinct from necrosis. Condensed, dying apoptotic cells, have been found in proliferative areas of tumour that are not under physiological stress. The analysis of apoptosis indicated a previously unsuspected variation in apoptotic activity with tumour weight. Cell death by apoptosis initially rose as the tumour grew, but after the tumour reached a threshold weight it declined dramatically, and finally remained stable. This may reflect an initial attempt at autoregulation of tumour size which ultimately fails. Apoptosis was estimated to account for an average of 7% of the total cell loss rate in this tumour.

Gut pathology of rats dosed with tetrathiomolybdate

Pathological changes occurred in the alimentary canal of rats fed a diet containing 3 mg per kg Cu and 6 mg per kg Mo as tetrathiomolybdate. Mitochondrial abnormalities in the duodenum and jejunum and single cell deletion by apoptosis and necrosis in the caecum and colon were present from the first day of treatment until the end of the investigation which terminated on the 21st day. These were accompanied by cell proliferation, presumably compensatory in character. When the rats had been given thiomolybdate for 11 days or more, there was a marked increase in the incidence of apoptosis in the small intestine, and gross disorganization and oedema of the caecal mucosa. There were also changes in the bacterial population of the caecal contents, where the Gram negative population became more complex and was dominated by species with the morphological characteristics of Bacteroides. These changes, with the exception of the mitochondrial lesions, could not be attributed to secondary hypocuprosis; it is suggested that the aetiology of the cell toxicity may involve a block in sulphur metabolism, possibly complicated in the caecum by absorption of endotoxins.

Adrenocortical involution in rats during oestrus synchronisation with medroxyprogesterone

Daily treatment of female rats with medroxyprogesterone acetate in aqueous suspension resulted in adrenocortical atrophy. The doses given were those used for oestrus synchronisation. Intramuscular injections of 2-0 mg medroxyprogesterone acetate were used to investigate the atrophic process. Adrenocortical involution was associated with extensive single cell deletion (apoptosis). It is suggested that theses changes were due to suppression of pituitary ACTH secretion. The cytological changes support the concept that single cell death plays an important role in organ remodelling. Biochemical determinations of DNA, RNA, protein and dry matter, and histological examination, did not reveal significant changes in the liver.

Localization of acid phosphatase activity in the liver of pregnant rats.

In the liver of pregnant rats, fed ad libitum, there was an increase in acid phosphatase specific activity which occurred in two peaks, one at the 15th day and the other at the end of gestation. By light and electron microscopic histochemistry, the activity was found to be localized in parenchymal cell peribiliary dense bodies and also in phagosomes present in macrophages and parenchymal cells. There was an increase in liver wieght which reached a peak at the 17th day of gestation. Total DNA also rose to the 17th day; there was a high rate of cell division in the hepatic parenchyma at the 17th and 18th days of gestation. During this period single cell deletion by apoptosis was relatively frequent and in late pregnancy there was evidence of cell deletion by lysis. During pregnancy there was a slight increase in sinusoidal macrophages as a proportion of the total cell population but there did not appear to be significant changes in macrophage enzymic activity. It is suggested that the acid phosphatase activity present in macrophages makes a minor contribution to total liver activity, most of which is present in parenchymal cells. Acid phosphatase activity associated with single cell deletion appears to be quantitatively negligible. There was a direct relationship between total hepatic acid phosphatase activity and the numbers of peribiliary dense bodies, which were most numerous at the 15th day and at the end of gestation. It is suggested that these residual bodies contain products of detoxification processes and also cell structural elements resulting from enhanced liver metabolism and intracellular turnover during pregnancy.