Properties Of Embryonic Stem Cells Research Articles

Abstract 294: Sox2 remodels the tumor microenvironment via recruitment of Tregs

Abstract Recent study showed that Sox2 gene, a key stem cell gene that maintains the pluripotency properties of embryonic stem cell (ESC) and cancer stem cell (CSC) plays an important role in the tumorigenesis and metastasis process of various types of tumor, however its regulatory effect on tumor microenvironment (TME) is still elusive. In this study, we established a BALB/c mice model xenografted with a mice breast cancer cell line-4T1 with down-regulation of Sox2 gene (4T1-shRNA-Sox2). We found that down-regulation of Sox2 inhibits the growth of tumors with a modulated immunomicroenvironment, which was reflected by the increased number of CD3+CD8+ T cells and expression level of Th1 cytokines, such as TFN-γ and decreased number of Foxp3+ Tregs and expression level of Th2 cytokines such as IL4, IL6 and IL10. In addition, the reduced expression level of vascular endothelial cell marker CD31 and lymphatic vessel marker Lyve-1 was also revealed in the 4T1-shRNA-Sox2 xenografted tumors. We further demonstrated that Sox2 modulates the TME by recruiting Tregs. By using real-time RT-PCR, we found the cytokines contributing to recruitment of Tregs, such as CCL1, CCL22, CCL28 and Cxcl12 reduced after Sox2 downregulation. In addition, the mRNA expression level of transcriptional factor NF-κB and its co-transcriptional factor RelA which controlled the expression of CCL1, CCL22 and CCL28 were also reduced. Consequently, our study revealed the functional interaction between tumor cells and the immunocytes in TME by demonstrated that Sox2 gene in tumor cells recruited Tregs to influence the tumor microenvironment thus promotes the growth of tumor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 294. doi:1538-7445.AM2012-294

The novel expression of Oct3/4 and Bmi1 in the root development of mouse molars

The root apex of the tooth elongates until the completion of root development. Although the signaling molecules inducing root elongation have been studied, the characteristic of the cells having the ability to maintain the root elongation remains unclear. This study aimed to investigate the characteristics of the cells involved in the root elongation. Octamer-binding factor 3/4 (Oct3/4) is known as one of the key regulators in maintaining the pluripotency and self-renewal properties of embryonic stem cells. Bmi1, the polycomb-group transcriptional repressor, has emerged as a key regulator in several cellular processes including stem cell self-renewal and cancer cell proliferation. At the beginning of root formation, ameloblasts expressed Oct3/4 in the nucleus, except in the apex of the cervical loop, in which Bmi1and cyclinD were expressed. At PN6, the expression of Oct3/4 in the ameloblasts shifted from the nucleus to the cytoplasm, whereas ameloblastin-negative Hertwig's epithelial root sheath (HERS) cells expressed Bmi1 and cyclinD. By PN10, the cells in the apex of HERS began to express Oct3/4 in their nucleus, whereas Bmi1 and cyclinD began to decrease in their expressions. The odontoblasts consistently expressed Oct3/4 in their cytoplasm. Our results suggest that (1) Oct3/4 creates the border between the ameloblasts from the proliferative region of HERS, (2) Bmi1-positive cells would be one of the candidates resulting in root elongation and (3) the Oct3/4 expression in the cytoplasm of odontoblasts may be related to maintain the odontoblastic characteristics.

LINE-1 Based Insertional Mutagenesis Screens to Identify Genes Involved in Embryonic Stem Cell Differentiation

Background: Knowledge of the intrinsic properties of embryonic stem cells is essential before the possibility of using these cells for therapeutic purposes becomes a reality. Insertional mutagenesis screens are widely used to identify genes sufficient to confer a particular phenotype; however, applications of such approaches are limited to mouse models. Thus, there is a need to develop a new DNA transposon system that allows gene discovery approaches in stem cells. Methods: This study was performed to explore the possibility of using the long interspersed nuclear element 1 (LINE -1) retrotransposon as a gene trap vector to identify genes involved in the maintenance and differentiation of mouse embryonic stem cells. Results: We developed an episomal, nonviral LINE-1 retrotransposon system using the scaffold/matrix attachment regions in the backbone of our vector. This gene trap vector harbors a GFP marker whose expression occurs only after successful insertional mutagenesis. By utilizing this vector, coupled with GFP expression, we have successfully isolated four individual embryonic stem cell clones that display disrupted genes, including two known genes. We then confirmed the identity of these genes using an inverse PCR approach and verified their function in cell differentiation using shRNAs and undifferentiated markers of embryonic stem cells. Conclusions: The ease of using this insertional mutagen and the simplicity of identifying the cells with disrupted genes by GFP expression make this LINE-1 vector a promising tool for embryonic stem cell and cancer stem cell gene discovery.

Amniotic Fluid and Amniotic Membrane Stem Cells: Marker Discovery

Amniotic fluid (AF) and amniotic membrane (AM) have been recently characterized as promising sources of stem or progenitor cells. Both not only contain subpopulations with stem cell characteristics resembling to adult stem cells, such as mesenchymal stem cells, but also exhibit some embryonic stem cell properties like (i) expression of pluripotency markers, (ii) high expansion in vitro, or (iii) multilineage differentiation capacity. Recent efforts have been focused on the isolation and the detailed characterization of these stem cell types. However, variations in their phenotype, their heterogeneity described by different groups, and the absence of a single marker expressed only in these cells may prevent the isolation of a pure homogeneous stem cell population from these sources and their potential use of these cells in therapeutic applications. In this paper, we aim to summarize the recent progress in marker discovery for stem cells derived from fetal sources such as AF and AM, using novel methodologies based on transcriptomics, proteomics, or secretome analyses.

Reality-Based Real-Time Cell Indentation Simulator

Training simulators that provide realistic visual and haptic feedback during cell indentation tasks are currently investigated. Complex cell geometry inherent to biological cells and intricate mechanical properties drive the need for precise mechanical and numerical modeling to assure accurate cell deformation and force calculations. Advances in alternative finite-element formulation, such as the mass-tensor approach, have reached a state, where they are applicable to model soft-cell deformation in real time. The geometrical characteristics and the mechanical properties of different cells are determined with atomic force microscopy (AFM) indentation. A real-time, haptics-enabled simulator for cell centered indentation has been developed, which utilizes the AFM data (mechanical and geometrical properties of embryonic stem cells) to accurately replicate the indentation task and predict the cell deformation during indentation in real time. This tool can be used as a mechanical marker to characterize the biological state of the cell. The operator is able to feel the change in the stiffness during cell deformation between fixed and live cells in real time. A comparative study with finite-element simulations using a commercial software and the experimental data demonstrate the effectiveness of the proposed physically based model.

Nucleolin Maintains Embryonic Stem Cell Self-renewal by Suppression of p53 Protein-dependent Pathway

Embryonic stem cells (ESCs) can undergo unlimited self-renewal and retain pluripotent developmental potential. The unique characteristics of ESCs, including a distinct transcriptional network, a poised epigenetic state, and a specific cell cycle profile, distinguish them from somatic cells. However, the molecular mechanisms underlying these special properties of ESCs are not fully understood. Here, we report that nucleolin, a nucleolar protein highly expressed in undifferentiated ESCs, plays an essential role for the maintenance of ESC self-renewal. When nucleolin is knocked down by specific short hairpin RNA (shRNA), ESCs display dramatically reduced cell proliferation rate, increased cell apoptosis, and G(1) phase accumulation. Down-regulation of nucleolin also leads to evident ESC differentiation as well as decreased self-renewal ability. Interestingly, expression of pluripotency markers (Oct4 and Nanog) is unaltered in these differentiated cells. Mechanistically, depletion of nucleolin up-regulates the p53 protein level and activates the p53-dependent pathway, at least in part, via increasing p53 protein stability. Silencing of p53 rescues G(1) phase accumulation and apoptosis caused by nucleolin deficiency entirely, although it partially blocks abnormal differentiation in nucleolin-depleted ESCs. It is noteworthy that knocking down nucleolin in NIH3T3 cells affected cell survival and proliferation in a much milder way, despite the comparable silencing efficiency obtained in ESCs and NIH3T3 cells. Collectively, our data demonstrate that nucleolin is a critical regulator of ESC self-renewal and that suppression of the p53-dependent pathway is the major molecular mechanism underlying functions of nucleolin in ESCs.

Abstract 9595: Generation of Transgene-Free Induced Pluripotent Stem Cells from Fibroblasts by Small Molecule Chemicals

Backgrounds: Reprogramming of somatic cells using viral- and non-viral vectors encoding defined transcription factors to generate induced pluripotent stem (iPS) cells has successfully been achieved over the past few years. However, the current established methods to create iPS cells require viral vectors or plasmids to deliver transgenes into the cells. Methods and Results: To obviate the putative problems of genetic contamination in the cells for future clinical application, it is highly demanded to develop new methods to create “safer” iPS cells without any genetic modifications. Here, we for the first time demonstrate successful generation of iPS cell line from mouse fibroblasts with only combinations of small-molecule chemicals such as GSK-3 beta inhibitor, histone deacetylase inhibitor, and G9a histone methyltransferase inhibitor, which act as epigenetic modulators and self-renewal activators. These chemical induced pluripotent stem (ChemiPS) cells have almost the same properties of embryonic stem cells in the expression patterns of pluripotency-related markers, epigenetic status in promoter regions of Oct4 and Nanog, spontaneous differentiation into three germ layers by in vitro embryoid body formation, and formation of teratoma in vivo after injection into mice. Conclusion: This study provides the first evidence that sole chemical combinations can reprogram somatic cells, resulting in transgene-free iPS cells. This method will advance the use of iPS cells in clinical application and disease investigation.

Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo kinetics of transplanted cells. A non-invasive method to repetitively monitor transplanted stem cells in vivo would allow investigators to directly monitor stem cell transplants and identify successful or unsuccessful engraftment outcomes. A wide range of stem cells continues to be investigated for countless applications. This protocol focuses on 3 different stem cell populations: human embryonic kidney 293 (HEK293) cells, human mesenchymal stem cells (hMSC) and induced pluripotent stem (iPS) cells. HEK 293 cells are derived from human embryonic kidney cells grown in culture with sheared adenovirus 5 DNA. These cells are widely used in research because they are easily cultured, grow quickly and are easily transfected. hMSCs are found in adult marrow. These cells can be replicated as undifferentiated cells while maintaining multipotency or the potential to differentiate into a limited number of cell fates. hMSCs can differentiate to lineages of mesenchymal tissues, including osteoblasts, adipocytes, chondrocytes, tendon, muscle, and marrow stroma. iPS cells are genetically reprogrammed adult cells that have been modified to express genes and factors similar to defining properties of embryonic stem cells. These cells are pluripotent meaning they have the capacity to differentiate into all cell lineages. Both hMSCs and iPS cells have demonstrated tissue regenerative capacity in-vivo. Magnetic resonance (MR) imaging together with the use of superparamagnetic iron oxide (SPIO) nanoparticle cell labels have proven effective for in vivo tracking of stem cells due to the near microscopic anatomical resolution, a longer blood half-life that permits longitudinal imaging and the high sensitivity for cell detection provided by MR imaging of SPIO nanoparticles. In addition, MR imaging with the use of SPIOs is clinically translatable. SPIOs are composed of an iron oxide core with a dextran, carboxydextran or starch surface coat that serves to contain the bioreactive iron core from plasma components. These agents create local magnetic field inhomogeneities that lead to a decreased signal on T2-weighted MR images. Unfortunately, SPIOs are no longer being manufactured. Second generation, ultrasmall SPIOs (USPIO), however, offer a viable alternative. Ferumoxytol (FerahemeTM) is one USPIO composed of a non-stoichiometric magnetite core surrounded by a polyglucose sorbitol carboxymethylether coat. The colloidal, particle size of ferumoxytol is 17-30 nm as determined by light scattering. The molecular weight is 750 kDa, and the relaxivity constant at 2T MRI field is 58.609 mM(-1) sec(-1) strength. Ferumoxytol was recently FDA-approved as an iron supplement for treatment of iron deficiency in patients with renal failure. Our group has applied this agent in an "off label" use for cell labeling applications. Our technique demonstrates efficient labeling of stem cells with ferumoxytol that leads to significant MR signal effects of labeled cells on MR images. This technique may be applied for non-invasive monitoring of stem cell therapies in pre-clinical and clinical settings.

High-throughput RNAi Screening Identifies a Role for TNK1 in Growth and Survival of Pancreatic Cancer Cells

To identify novel targets in pancreatic cancer cells, we used high-throughput RNAi (HT-RNAi) to select genes that, when silenced, would decrease viability of pancreatic cancer cells. The HT-RNAi screen involved reverse transfecting the pancreatic cancer cell line BxPC3 with a siRNA library targeting 572 kinases. From replicate screens, approximately 32 kinases were designated as hits, of which 22 kinase targets were selected for confirmation and validation. One kinase identified as a hit from this screen was tyrosine kinase nonreceptor 1 (TNK1), a kinase previously identified as having tumor suppressor-like properties in embryonic stem cells. Silencing of TNK1 with siRNA showed reduced proliferation in a panel of pancreatic cancer cell lines. Furthermore, we showed that silencing of TNK1 led to increased apoptosis through a caspase-dependent pathway and that targeting TNK1 with siRNA can synergize with gemcitabine treatment. Despite previous reports that TNK1 affects Ras and NF-κB signaling, we did not find similar correlations with these pathways in pancreatic cancer cells. Our results suggest that TNK1 in pancreatic cancer cells does not possess the same tumor suppressor properties seen in embryonic cells but seems to be involved in growth and survival. The application of functional genomics by using HT-RNAi screens has allowed us to identify TNK1 as a growth-associated kinase in pancreatic cancer cells.

Dissecting the role of SALL4, a newly identified stem cell factor, in chronic myelogenous leukemia

Dissecting the role of SALL4, a newly identified stem cell factor, in chronic myelogenous leukemia

SOX2 promotes tumorigenesis and increases the anti-apoptotic property of human prostate cancer cell

SRY-related HMG-box gene 2 (SOX2) is one of the key regulatory genes that maintain the pluripotency and self-renewal properties in embryonic stem cells. Here we used immunohistochemistry to analyze the expression of SOX2 in human prostate tissues and found it contributed to tumorigenesis and correlated with histologic grade and Gleason score. We further investigated SOX2's function in cell growth and apoptosis process by using a human prostate cancer cell line DU145 with SOX2 overexpression or down-regulation. Cell cycle assay revealed that SOX2 promoted cell growth and increased the percentage of cells in S phase. In vitro and in vivo xenograft experiments in NOD/SCID mice further demonstrated that SOX2 increased the apoptosis-resistant properties of DU145 cells with decreased function of store-operated Ca(2+) entry and reduced expression of Orai1 at both mRNA and protein levels, suggesting a potential mechanism that contributes to the anti-apoptotic property of SOX2. To our knowledge, this study is the first to investigate SOX2's function in tumorigenesis and apoptosis of human prostate cancer and to elucidate its regulatory effect on the activity of store-operated Ca(2+) channels. Our results support the concept that SOX2 has the potential to be a significant marker to evaluate the progression of prostate cancer and serve as a potentially useful target for prostate cancer therapy.

Retinol (vitamin A) maintains self‐renewal of pluripotent male germline stem cells (mGSCs) from adult mouse testis

Studies have shown that male germline stem cells (mGSCs), which are responsible for maintaining spermatogenesis in the male, could be obtained from mouse and human testis. However, the traditional cultural methods were mostly dependent on serum and feeder, and the initial mGSCs were either obtained from neonatal mice or the detailed description of its potency and origin was not provided. Here we reported a novel (retinol (RE) serum-free and feeder-free) system for the successful culture of adult germline stem cells from adult Kunming mice (8-24 weeks) testis. The isolated mGSCs cultured in RE serum-free and feeder-free medium maintained the typical morphology of undifferentiated embryonic stem cells (ESCs), and they proliferated well in RE medium analyzed by proliferation assay, RT-PCR, microarray, and Western blotting. These cells also showed typical properties of ESCs (alkaline phosphatase (AP) positive, expressions of Oct4, Sox2, Nanog, and SSEA1, with the capacity to form teratomas and differentiate into various types of cells within three germ layers). Taken together, we conclude that RE promotes the self-renewal of mGSCs and maintains the pluripotency of mGSCs, the RE serum-free and feeder-free system may be useful for the culture of pluripotent stem cell lines from adult testis tissues, which provides a new resource for tissue engineering and therapy for infertility.

Dual Functions of T-Box 3 (Tbx3) in the Control of Self-renewal and Extraembryonic Endoderm Differentiation in Mouse Embryonic Stem Cells

Embryonic stem cells (ESCs) possess the capacity to proliferate indefinitely in an undifferentiated state and to differentiate into various cell types in an organism. However, the critical question of how self-renewal and differentiation are precisely regulated in ESCs is not entirely understood at present. Here, we report the essential role of Tbx3, a pluripotency-related transcription factor of the T-box gene family, for both the maintenance of self-renewal of mouse ESCs and for their differentiation into extraembryonic endoderm (ExEn). We show that Tbx3 is highly expressed in ExEn cells in addition to undifferentiated ESCs. Knockdown of Tbx3 expression using tetracycline-regulated Tbx3 siRNA resulted in the attenuation of ESC self-renewal ability and aberrant differentiation processes, including reduced ExEn differentiation but enhanced ectoderm and trophectoderm differentiation. Conversely, inducible forced expression of Tbx3 triggered ExEn lineage commitment. Mechanistically, Tbx3 directly activated the expression of Gata6, an essential regulator of ExEn. Interestingly, Tbx3 modulated H3K27me3 modification and the association of the PRC2 complex with the promoter region of Gata6. Taken together, the results of this study revealed a previously unappreciated role of a pluripotency factor in ExEn differentiation. Additionally, our data reveal that Tbx3 may function through direct binding and epigenetic modification of histones on the Gata6 promoter to maintain the ExEn differentiation potential of ESCs.

Endogenous Expression of Matriptase in Neural Progenitor Cells Promotes Cell Migration and Neuron Differentiation

Recent studies show that type II transmembrane serine proteases play important roles in diverse cellular activities and pathological processes. Their expression and functions in the central nervous system, however, are largely unexplored. In this study, we show that the expression of one such member, matriptase (MTP), was cell type-restricted and primarily expressed in neural progenitor (NP) cells and neurons. Blocking MTP expression or MTP activity prevented NP cell traverse of reconstituted basement membrane, whereas overexpression of MTP promoted it. The NP cell mobilization induced by either vascular endothelial growth factor or hepatocyte growth factor was also impaired by knocking down MTP expression. MTP acts upstream of matrix metalloproteinase 2 in promoting NP cell mobility. In embryonic stem cell differentiation to neural cells, MTP knockdown had no effect on entry of embryonic stem cells into the neural lineage. High MTP expression or activity, however, shifts the population dynamics from NP cells toward neurons to favor neuronal differentiation. This is the first report to demonstrate the direct involvement of type II transmembrane serine protease in NP cell function.

Landmark Approach to Generating Human Stem Cells

Building on the breakthrough discovery that differentiated cells can be returned to a pluripotent state, a team of researchers led by Dr. Derrick Rossi of Harvard Medical School have developed a powerful new technique for cellular reprogramming that could have a broad range of applications in both clinical and research settings. Ongoing ethical concerns about the study of embryonic stem cells for the treatment of disease propelled researchers to search for methods to generate pluripotent human stem cells in the laboratory. The first milestone was reported in 2006 by a team of investigators led by Shinya Yamanaka of Kyoto University. Their research focused on 24 select genes believed to contribute to the regulation of embryonic stem cell properties. After screening each of the candidate genes in cultures of mouse embryonic and adult cells, they showed that only four were sufficient to induce pluripotency. The genes encode transcription factors identified as Klf4, c-Myc, Oct4, and Sox2, and the reprogrammed cells were termed induced pluripotent stem (iPS) cells.1 In a subsequent investigation, Yamanaka and colleagues studied the effects of the same four genes in fully differentiated, adult human dermal fibroblasts. By using retroviruses to incorporate the genes into the fibroblast DNA, the researchers forced the coexpression of the four transcription factors and reverted the mature cells back to a pluripotent state. The result was the first iPS cells made from mature, differentiated human cells.2 Yamanaka's research marked a major turning point in cellular reprogramming; however, his technique was not without important limitations. First, it was inefficient, with an approximate yield of only 10 iPS colonies from 50,000 transduced human fibroblasts. In addition, the first colonies resembling human embryonic cells did not appear for about …

Chromatin structure of pluripotent stem cells and induced pluripotent stem cells

Pluripotent embryonic stem (ES) cells are specialized cells with a dynamic chromatin structure, which is intimately connected with their pluripotency and physiology. In recent years somatic cells have been reprogrammed to a pluripotent state through over-expression of a defined set of transcription factors. These cells, known as induced pluripotent stem (iPS) cells, recapitulate ES cell properties and can be differentiated to apparently all cell lineages, making iPS cells a suitable replacement for ES cells in future regenerative medicine. Chromatin modifiers play a key function in establishing and maintaining pluripotency, therefore, elucidating the mechanisms controlling chromatin structure in both ES and iPS cells is of utmost importance to understanding their properties and harnessing their therapeutic potential. In this review, we discuss recent studies that provide a genome-wide view of the chromatin structure signature in ES cells and iPS cells and that highlight the central role of histone modifiers and chromatin remodelers in pluripotency maintenance and induction.

Role of the Epigenetic Regulator HP1γ in the Control of Embryonic Stem Cell Properties

The unique properties of embryonic stem cells (ESC) rely on long-lasting self-renewal and their ability to switch in all adult cell type programs. Recent advances have shown that regulations at the chromatin level sustain both ESC properties along with transcription factors. We have focused our interest on the epigenetic modulator HP1γ (Heterochromatin Protein 1, isoform γ) that binds histones H3 methylated at lysine 9 (meH3K9) and is highly plastic in its distribution and association with the transcriptional regulation of specific genes during cell fate transitions. These characteristics of HP1γ make it a good candidate to sustain the ESC flexibility required for rapid program changes during differentiation. Using RNA interference, we describe the functional role of HP1γ in mouse ESC. The analysis of HP1γ deprived cells in proliferative and in various differentiating conditions was performed combining functional assays with molecular approaches (RT-qPCR, microarray). We show that HP1γ deprivation slows down the cell cycle of ESC and decreases their resistance to differentiating conditions, rendering the cells poised to differentiate. In addition, HP1γ depletion hampers the differentiation to the endoderm as compared with the differentiation to the neurectoderm or the mesoderm. Altogether, our results reveal the role of HP1γ in ESC self-renewal and in the balance between the pluripotent and the differentiation programs.

Influence of cocultivation with vital and ischemic ventricular tissue on electrophysiological properties of embryonic stem cell–derived cardiomyocytes

Influence of cocultivation with vital and ischemic ventricular tissue on electrophysiological properties of embryonic stem cell–derived cardiomyocytes

Tumorigenicity of pluripotent stem cells: biological insights from molecular imaging

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have the ability (i) to duplicate indefinitely while maintaining pluripotency and (ii) to differentiate into cell types of all three embryonic germ layers. These two properties of ESCs and iPSCs make them potentially suitable for tissue engineering and cell replacement therapy for many different diseases, including Parkinson's disease, diabetes and heart disease. However, one critical obstacle in the clinical application of ESCs or iPSCs is the risk of teratoma formation. The emerging field of molecular imaging is allowing researchers to track transplanted ESCs or iPSCs in vivo, enabling early detection of teratomas.

Molecular Basis of Mammalian Embryonic Stem Cell Pluripotency and Self-Renewal

Mammalian embryonic stem cells (ESC) have a number of specific properties that make them a unique object of fundamental and applied studies. In culture, ESC can remain in an infinitely undifferentiated state and differentiate into descendants of all three germ layers - ectoderm, endoderm, and mesoderm - that is, they can potentially produce more than 200 cell types comprising the body of an adult mammal. These properties of ESC are refered to as self-renewal and pluripotency. In this review, the basic signal pathways implicated in the maintenance of ESC pluripotency are considered. The major genes comprising a subsystem of "internal regulators of pluripotency," their protein products and regulators, are characterized, and interaction with other factors is described as well. The role of epigenetic mechanisms and microRNAs in the system of ESC self-renewal and pluripotency, as well as the relationship between pluripotency and X-chromosome inactivation in female mammals, is discussed.