Expression Of Cell Growth Research Articles

New Insights into the Roles of the Hippo Signaling Pathway in Stem Cell Biology

: A highly conserved kinase cascade, including mammalian STE20-like protein kinases (MSTs), large tumor suppressors (LATSs), and downstream transcription coactivators containing YES-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are involved in the Hippo signaling pathway. As two critical transcriptional coactivators, TAZ and YAP are adversely regulated by the Hippo signaling pathway. They lead to the induction of organ regeneration and the expression of cell growth and division-promoting genes through binding to transcription factors, especially TEA domain transcription factors (TEADs). Previous studies highlight the role of the Hippo pathway in organ size control, homeostasis, cell proliferation, and tumor development. Research has shown this pathway's vital role in cancer stem cell biology, including self-renewal and drug resistance. Improper activation of YAP and TAZ through the Hippo pathway dysregulation or its increased expression can accelerate tumorigenesis. Thus, pharmacological inhibition of YAP and TAZ is suggested as a promising approach in the treatment of tumors with high YAP/TAZ activity.

Focused Low-Intensity Pulsed Ultrasound (FLIPUS) Mitigates Apoptosis of MLO-Y4 Osteocyte-like Cells

Long cytoplasmic processes of osteocytes orchestrate bone activity by integration of biochemical and mechanical signals and regulate load-induced bone adaptation. Low-Intensity Pulsed Ultrasound (LIPUS) is a clinically used technique for fracture healing that delivers mechanical impulses to the damaged bone tissue in a non-invasive and non-ionizing manner. The mechanism of action of LIPUS is still controversially discussed in the scientific community. In this study, the effect of focused LIPUS (FLIPUS) on the survival of starved MLO-Y4 osteocytes was investigated in vitro. Osteocytes stimulated for 10 min with FLIPUS exhibited extended dendrites, which formed frequent connections to neighboring cells and spanned longer distances. The sonicated cells displayed thick actin bundles and experienced increase in expression of connexin 43 (Cx43) proteins, especially on their dendrites, and E11 glycoprotein, which is responsible for the elongation of cellular cytoplasmic processes. After stimulation, expression of cell growth and survival genes as well as genes related to cell-cell communication was augmented. In addition, cell viability was improved after the sonication, and a decrease in ATP release in the medium was observed. In summary, FLIPUS mitigated apoptosis of starved osteocytes, which is likely related to the formation of the extensive dendritic network that ensured cell survival.

Metabolic regulation of ethanol-type fermentation of anaerobic acidogenesis at different pH based on transcriptome analysis of Ethanoligenens harbinense

BackgroundEthanol-type fermentation, one of the fermentation types in mixed cultures of acidogenesis with obvious advantages such as low pH tolerance and high efficiency of H2 production, has attracted widespread attentions. pH level greatly influences the establishment of the fermentation of carbohydrate acidogenesis by shaping community assembly and the metabolic activity of keystone populations. To explore the adaptation mechanisms of ethanol-type fermentation to low pH, we report the effects of initial pH on the physiological metabolism and transcriptomes of Ethanoligenens harbinense—a representative species of ethanol-type fermentation.ResultsDifferent initial pH levels significantly changed the cell growth and fermentation products of E. harbinense. Using transcriptomic analysis, we identified and functionally categorized 1753 differentially expressed genes (DEGs). By mining information on metabolic pathways, we probed the transcriptional regulation of ethanol–H2 metabolism relating to pH responses. Multiple pathways of E. harbinense were co-regulated by changing gene expression patterns. Low initial pH down-regulated the expression of cell growth- and acidogenesis-related genes but did not affect the expression of H2 evolution-related hydrogenase and ferredoxin genes. High pH down-regulated the expression of H2 evolution- and acidogenesis-related genes. Multiple resistance mechanisms, including chemotaxis, the phosphotransferase system (PTS), and the antioxidant system, were regulated at the transcriptional level under pH stress.ConclusionsEthanoligenens adapted to low pH by regulating the gene expression networks of cell growth, basic metabolism, chemotaxis and resistance but not H2 evolution-related genes. Regulation based on pH shifts can represent an important approach to establish and enhance ethanol-type fermentation. The complete gene expression network of ethanol fermentative bacteria for pH response provides valuable insights into the acidogenic fermentation, and offers an effective regulation strategy for the sustainable energy recovery from wastewater and solid waste.

Early thinning of peach fruit influences expression of cell growth and expansion genes

Early thinning of peach fruit influences expression of cell growth and expansion genes

In vitro study on siRNA integrin α_vβ_3 gene expression inhibiting glioma U251 cells proliferation

Objective Explore the specificity of siRNA interference targeting malignant glioma U251 cells, INTα_vβ_3 expression of cell growth after the role. Method U251 cells were transfected with INT α_vβ_3 specific siRNA by lipofectamine(LipefectamineTM~(2000)). RT-PCR, immune ceils staining and Western blot were used to determine the expression of INTα_vβ_3. Cell proliferation after the detection of changes in interference was tested by MTT. Results INTα_vβ_3 specific siRNA shown to inhibit U251 cell proliferation. There were statistical significance between siRNA group, liposome group and the control group at different time points. Green INTα_vβ_3 expression was found in control group and lipesome groups, and reduced in siRNA group. Western blotting and RT-PCR results shown that the expression of integrin could be inhibited by α_vβ_3 transfection in the protein and mRNA level Conclusions Integrin-specific siRNA INTα_vβ_3 can inhibit the U251 glioma cell proliferation. Key words: siRNA; Integrin α_vβ_3; Glioma; 13251 cells

Effect of high cell density on the growth properties of tumor cells: a role in tumor cytotoxicity of chemotherapeutic drugs

The aim of this study was to understand the role of tumor progression in the growth properties of tumor cells and their susceptibility to the cytotoxicity of chemotherapeutic drugs. A murine transplantable T cell lymphoma of spontaneous origin, designated as Dalton's lymphoma, was used as a model tumor for this investigation. Tumor cells were harvested from the early (5 days after tumor transplantation) and late tumor-bearing stages (17 days after tumor transplantation), with or without in-vivo administration of the chemotherapeutic drugs, cisplatin or doxorubicin. Tumor cells harvested at the late tumor-bearing stages showed a higher proliferative ability in vitro. Tumor progression was found to be associated with a decline in the tumor cytotoxicity of the chemotherapeutic drugs. Similar results were also obtained when tumor cells were cultured at low (10(5) cells/ml) and high (10(9) cells/ml) cell densities in vitro in medium alone or in one containing the chemotherapeutic drugs. An increase in the expression of heat shock protein (Hsp70), vascular endothelial growth factor, interleukin-2 receptor and interleukin-2 proteins along with an inhibition in the expression of caspase-activated DNase and p53 proteins was observed during the late tumor-bearing stage and also in the Dalton's lymphoma cells when cultured in vitro at a higher cell density. The ascitic fluid obtained from the late tumor-bearing stage and the culture supernatant of tumor cells incubated in vitro at high cell density showed high levels of cell growth-regulating cytokines: interleukin-1, interleukin-2, interferon-gamma, vascular endothelial growth factor, tumor growth factor-beta and interleukin-10. In-vivo administration of cisplatin in tumor-bearing mice at the late tumor-bearing stage did not alter the level of these cytokines in the ascitic fluid. In view of the results of this investigation, it is suggested that under high cellular density-associated environmental conditions the tumor cells alter their growth properties depending on an alteration in the expression of cell growth and apoptosis-regulating proteins. Tumor cells, thus, switch to a high level of proliferation, which renders them resistant to the cytotoxicity of chemotherapeutic drugs.

A Role for DNA Methylation in Regulating the Growth Suppressor PMEPA1 Gene in Prostate Cancer

A cascade of epigenetic events contributes to the selective growth advantage of cancer cells during tumor progression. PMEPA1 gene is an androgen-inducible negative regulator of cell growth in the prostate epithelium. During prostate cancer progression PMEPA1 gene transcription is reduced or lost prompting us to investigate the role of epigenetic events in this process. In LAPC4 cells harboring wild type androgen receptor decitabine (5-aza-2’-deoxycitidine) treatment resulted in increased expression of PMEPA1 along with other androgen-inducible genes suggesting a role for DNA methylation in the repression of androgenic cell growth control signals in prostate cancer. In contrast, mutant androgen receptor expressing LNCaP cells were deficient in this response. Therefore, decitabine-induced expression of cell growth controlling genes such as NKX3.1 or PMEPA1 underlines the clinical applicability of decitabine in prostate tumors harboring wild type androgen receptor. Further analysis of DNA methylation within the PMEPA1 promoter downstream sequences suggests that methylation of SP1 binding sites may also contribute to the repression of PMEPA1 gene.

Gene expression profile of butyrate-inhibited vascular smooth muscle cell proliferation.

Excessive proliferation of vascular smooth muscle cells (VSMCs) is a critical element in the development of several vascular pathologies, particularly in atherosclerosis and in restenosis due to angioplasty. We have shown that butyrate, a powerful antiproliferative agent, a strong promoter of cell differentiation and an inducer of apoptosis inhibits VSMC proliferation at physiological concentrations with no cytotoxicity. In the present study, we have used cDNA array technology to unravel the molecular basis of the antiproliferative effect of butyrate on VSMCs. To assess the involvement of gene expression in butyrate-inhibited VSMC proliferation, proliferating VSMCs were exposed to 5 mmol/l butyrate 1 through 5 days after plating. Expression profiles of 1.176 genes representing different functional classes in untreated control and butyrate treated VSMCs were compared. A total of 111 genes exhibiting moderate (2.0-5.0 fold) to strong (> 5.0 fold) differential expression were identified. Analysis of these genes indicates that butyrate treatment mainly alters the expression of four different functional classes of genes, which include: 43 genes implicated in cell growth and differentiation, 13 genes related to stress response, 11 genes associated with vascular function and 8 genes normally present in neuronal cells. Examination of differentially expressed cell growth and differentiation related genes indicate that butyrate-inhibited VSMC proliferation appears to involve down-regulation of genes that encode several positive regulators of cell growth and up-regulation of some negative regulators of growth or differentiation inducers. Some of the down-regulated genes include proliferating cell nuclear antigen (PCNA), retinoblastoma susceptibility related protein p130 (pRb), cell division control protein 2 homolog (cdc2), cyclin B1, cell division control protein 20 homolog (p55cdc), high mobility group (HMG) 1 and 2 and several others. Whereas the up-regulated genes include cyclin D1, p21WAF1, p141NK4B/p15INK5B, Clusterin, inhibitor of DNA binding 1 (ID1) and others. On the other hand, butyrate-responsive stress-related genes include some of the members of heat shock protein (HSP), glutathione-s-transferase (GST), glutathione peroxidase (GSH-PXs) and cytochrome P450 (CYP) families. Additionally, several genes related to vascular and neuronal function are also responsive to butyrate treatment. Although involvement of genes that encode stress response, vascular and neuronal functional proteins in cell proliferation is not clear, cDNA expression array data appear to suggest that they may play a role in the regulation of cell proliferation. However, cDNA expression profiles indicate that butyrate-inhibited VSMC proliferation involves combined action of a proportionally large number of both positive and negative regulators of growth, which ultimately causes growth arrest of VSMCs. Furthermore, these butyrate-induced differential gene expression changes are not only consistent with the antiproliferative effect of butyrate but are also in agreement with the roles that these gene products play in cell proliferation.

Species-Specific Glucocorticoid and 1,25-Dihydroxyvitamin D Responsiveness in Mouse MC3T3-E1 Osteoblasts: Dexamethasone Inhibits Osteoblast Differentiation and Vitamin D Down-Regulates Osteocalcin Gene Expression*

The mouse MC3T3-E1 cell line is nontumorigenic and undergoes a typical program of osteoblast differentiation in vitro, producing a bone-like mineralized extracellular matrix. We report responses of these cells to dexamethasone (Dex) and 1,25-(OH)2D3 that are in contrast to findings from other osteoblast culture systems. First, chronic exposure of both early- and late-passaged MC3T3-E1 cells to 10(-7) M Dex, initiated during the proliferation period, blocked osteoblast differentiation, in contrast to the enhanced differentiation observed in cultures of fetal rat calvarial-derived cells. Secondly, 1,25-(OH)2D3 did not up-regulate expression (messenger RNA or protein synthesis) of the endogenous mouse osteocalcin (OC) gene. Several lines of evidence are presented that suggest this response is caused by sequence specific properties of the mouse OC vitamin D response element. We also observed both qualitative and quantitative differences in expression of cell growth (histone H2B) and phenotype-related genes (collagen, OC, osteopontin, glucocorticoid receptor, and 1, 25-(OH)2D3 receptor), between pre- and postmineralization stage osteoblasts, in response to 24 h steroid hormone treatment. Our findings in MC3T3-E1 cells are consistent with current concepts of selective influences of 1,25-(OH)2D3 and glucocorticoids as a function of osteoblast maturation. However, the inhibition of osteoblast differentiation by chronic Dex at 10(-7) M and the down-regulation of OC by 1,25-(OH)2D3 are novel observations relevant to species-specific responsiveness of mouse bone-expressed genes to steroid hormones during osteoblast differentiation.

Acidic fibroblast growth factor inhibits osteoblast differentiation in vitro: Altered expression of collagenase, cell growth-related, and mineralization-associated genes

Fibroblast growth factors (FGF) are osteoblast mitogens, but their effects on bone formation are not clearly understood. Most in vitro studies examining the effects of FGFs on osteoblasts have been performed only during the initial proliferative stage of osteoblast culture. In these studies, we examined the consequential effect of acidic FGF in cultures of rat fetal diploid osteoblasts that undergo a developmental differentiation program producing a mineralized bone-like matrix. During the initial growth period (days 1-10), addition of acidic FGF (100 micrograms/ml) to actively proliferating cells increased (P < 0.05) 3H-thymidine uptake (2,515 +/- 137, mean +/- SEM vs. 5,884 +/- 818 cpm/10(4) cells). During the second stage of maturation (days 10-15), osteoblasts form multilayered nodules of cells and accumulate matrix, followed by mineralization (stage 3, days 16-29). Addition of acidic FGF to the osteoblast cultures from days 7 to 15 completely blocked nodule formation. Furthermore, addition of acidic FGF after nodule formation (days 14-29) inhibited matrix mineralization, which was associated with a marked increase in collagenase gene expression, and resulted in a progressive change in the morphology of the nodules, with only a few remnants of nonmineralized nodules present by day 29. Histochemical and biochemical analyses revealed a decrease in alkaline phosphatase and mineral content, confirming the acidic FGF-induced inhibition of nodule and matrix formation. To identify mechanisms contributing to these changes, we examined expression of cell growth and bone phenotypic markers. Addition of acidic FGF during the proliferative phase (days 7-8) enhanced histone H4, osteopontin, type I collagen, and TGF-beta mRNA levels, which are coupled to proliferating osteoblasts, and blocked the normal developmental increase in alkaline phosphatase and osteocalcin gene expression and calcium accumulation. Addition of acidic FGF to the cultures during matrix maturation (days 14-15) reactivated H4, osteopontin, type I collagen, and TGF-beta gene expression, and decreased alkaline phosphatase and osteocalcin gene expression. In an in vivo experiment, rats were treated with up to 60 micrograms/kg/day acidic FGF intravenously for 30 days. Proliferation of osteoblasts and deposition of bone occurred in the marrow space of the diaphysis of the femur in a dose-related fashion. The metaphyseal areas were unaffected by treatment. In conclusion, our data suggest that acidic FGF is a potent mitogen for early stage osteoblasts which leads to modifications in the formation of the extracellular matrix; increases in TGF-beta and collagenase are functionally implicated in abrogating competency for nodule formation. Persistence of proliferation prevented expression of alkaline phosphatase and osteocalcin, also contributing to the block in the progression of the osteoblast developmental sequence.

Expression of Cell Growth Regulated Genes in the Fetal Kidney

Expression of Cell Growth Regulated Genes in the Fetal Kidney

Expression of cell growth and bone phenotypic genes during the cell cycle of normal diploid osteoblasts and osteosarcoma cells

Establishing regulatory mechanisms that mediate proliferation of osteoblasts while restricting expression of genes associated with mature bone cell phenotypic properties to post-proliferative cells is fundamental to understanding skeletal development. To gain insight into relationships between growth control and the developmental expression of genes during osteoblast differentiation, we have examined expression of three classes of genes during the cell cycle of normal diploid rat calvarial-derived osteoblasts and rat osteosarcoma cells (ROS 17/2.8): cell cycle and growth-related genes (e.g., histone), genes that encode major structural proteins (e.g., actin and vimentin), and genes related to the biosynthesis, organization, and mineralization of the bone extracellular matrix (e.g., alkaline phosphatase, collagen I, osteocalcin, and osteopontin). In normal diploid osteoblasts as well as in osteosarcoma cells we found that histone genes, required for cell progression, are selectively expressed during S phase. All other genes studied were constitutively expressed both at the transcriptional and posttranscriptional levels. Alkaline phosphatase, an integral membrane protein in both osteoblasts and osteosarcoma cells, exhibited only minimal changes in activity during the osteoblast and osteosarcoma cell cycles. Our findings clearly indicate that despite the loss of normal proliferation-differentiation interrelationships in osteosarcoma cells, cell cycle regulation or constitutive expression of growth and phenotypic genes is maintained.

TGFβ alters growth and differentiation related gene expression in proliferating osteoblasts in vitro, preventing development of the mature bone phenotype

This study examines the mechanism by which TGF-beta 1, an important mediator of cell growth and differentiation, blocks the differentiation of normal rat diploid fetal osteoblasts in vitro. We have established that the inability for pre-osteoblasts to differentiate is associated with changes in the expression of cell growth, matrix forming, and bone related genes. These include histone, jun B, c-fos, collagen, fibronectin, osteocalcin, alkaline phosphatase, and osteopontin. Morphologically, the TGF-beta 1-treated osteoblasts exhibit an elongated, spread shape as opposed to the characteristic cuboidal appearance during the early stages of growth. This is followed by a decrease in the number of bone nodules formed and the amount of calcium deposition. These effects on differentiation can occur without dramatic changes in cell growth if TGF-beta 1 is given for a short time early in the proliferative phase. However, continuous exposure to TGF-beta 1 leads to a bifunctional growth response from a negative effect during the proliferative phase to a positive growth effect during the later matrix maturation and mineralization phases of the osteoblast developmental sequence. Extracellular matrix genes, fibronectin, osteopontin and alpha 1(I) collagen, are altered in their expression pattern which may provide an aberrant matrix environment for mineralization and osteoblast maturation and potentiate the TGF-beta 1 response throughout the course of osteoblast differentiation. The initiation of a TGF-beta 1 effect on cell growth and differentiation is restricted to the proliferative phase of the culture before the cells express the mature osteoblastic phenotype. Second passage cells that are accelerated to differentiate by the addition of dexamethasone or by seeding cultures at a high density are refractory to TGF-beta 1. These in vitro results indicate that TGF-beta 1 exerts irreversible effects at a specific stage of osteoblast phenotype development resulting in a potent inhibition of osteoblast differentiation at concentrations from 0.1 ng/ml.

Aberrant gene expression in cultured mammalian bone cells demonstrates an osteoblast defect in osteopetrosis

Osteopetrosis is a skeletal condition in which a generalized radioopacity of bone is caused by reduced resorption of bone by osteoclasts. However, it has recently been shown that during skeletal development in several osteopetrotic rat mutations specific aberrations occur in gene expression reflecting the activity of the bone forming cells, osteoblasts, and the development of tissue organization. To evaluate their pathogenetic significance, progressive osteoblast differentiation was studied in vitro. Primary cultures of normal osteoblasts undergo a sequential expression of cell growth and tissue-related genes associated with development of skeletal tissue. We report that osteoblast cultures can be established from one of these mutants, toothless; that these cells in vitro exhibit similar aberrations in gene expression during cell proliferation and extracellular matrix formation and mineralization observed in vivo; and that an accelerated maturation sequence by mutant osteoblasts mimics the characteristic skeletal sclerosis of this disease. These data are the first direct evidence for an intrinsic osteoblast defect in osteopetrosis and establish an in vitro model for the study of heritable skeletal disorders.

Influence of DNA replication inhibition on expression of cell growth and tissue-specific genes in osteoblasts and osteosarcoma cells.

Interrelationships between proliferation and expression of cell growth as well as bone cell-related genes were examined from two standpoints. First, the consequence of downregulating proliferation by DNA synthesis inhibition on expression of a cell cycle-regulated histone gene and genes associated with development of the bone cell phenotype (type I collagen, alkaline phosphatase, osteopontin, and osteocalcin) was investigated. Second, the requirement for stringent growth control to support functional relationships between expression of proliferation and differentiation-related genes was explored. Parameters of cell growth and osteoblast-related gene expression in primary cultures of normal diploid osteoblasts, that initially express proliferation-dependent genes and subsequently postproliferative genes associated with mature bone cell phenotypic properties, were compared to those operative in ROS 17/2.8 osteosarcoma cells that concomitantly express cell growth and mature osteoblast phenotypic genes. Our findings indicate that in both normal diploid osteoblasts and osteosarcoma cells, expression of the cell cycle regulated histone genes is tightly coupled with DNA synthesis and controlled predominantly at a posttranscriptional level. Inhibition of proliferation by blocking DNA synthesis with hydroxyurea upregulates a subset of developmentally expressed genes that postproliferatively support progressive establishment of mature osteoblast phenotypic properties (e.g., alkaline phosphatase, type 1 collagen, and osteopontin). However, the osteocalcin gene, which is expressed during the final stage of osteoblast differentiation when extracellular matrix mineralization occurs, is not upregulated. Variations in the extent to which inhibition of proliferation in normal diploid osteoblasts and in ROS 17/2.8 osteosarcoma cells selectively affects transcription and cellular levels of mRNA transcripts from bone cell-related genes (e.g., osteocalcin) may reflect modifications in proliferation/differentiation interrelationships when stringent growth control is abrogated.

Effects of 1,25(OH) 2D 3 and vitamin D analogs on developmental control of cell growth and tissue-specific gene expression during osteoblast differentiation

Cultured normal diploid osteoblasts provide a model for identifying selective effects of vitamin D and 1,25(OH) 2D 3 analogs on expression of cell growth and tissue-specific genes that are dependent on the differentiated state of the bone cell. Transcription of the bone-specific osteocalcin gene is responsive to the vitamin D receptor complex together with other cellular signaling factors. Cultured normal diploid osteoblasts provide a model for identifying selective effects of vitamin D and 1,25-(OH) 2D 3 analogs on expression of cell growth and tissue-specific genes that are dependent on the differentiated state of the bone cell. Transcription of the bone-specific osteocalcin gene is responsive to the vitamin D receptor complex together with other cellular signaling factors.

Expression of cell growth and bone specific genes at single cell resolution during development of bone tissue-like organization in primary cultures of normal diploid osteoblasts

Expression of cell growth and bone specific genes at single cell resolution during development of bone tissue-like organization in primary cultures of normal diploid osteoblasts

Expression of cell growth and bone specific genes at single cell resolution during development of bone tissue-like organization in primary osteoblast cultures.

Primary cultures of calvarial derived normal diploid osteoblasts undergo a developmental expression of genes reflecting growth, extracellular matrix maturation, and mineralization during development of multilayered nodules having a bone tissue-like organization. Scanning electron microscopy of the developing cultures indicates the transition from the uniform distribution of cuboidal osteoblasts to multilayered nodules of smaller cells with a pronounced orientation of perinodular cells towards the apex of the nodule. Ultrastructural analysis of the nodule by transmission electron microscopy indicates that the deposition of mineral is confined to the extracellular matrix where cells appear more osteocytic. The cell body contains rough endoplasmic reticulum and golgi, while these intracellular organelles are not present in the developing cellular processes. To understand the regulation of temporally expressed genes requires an understanding of which genes are selectively expressed on a single cell basis as the bone tissue-like organization develops. In situ hybridization analysis using 35S labelled histone gene probes, together with 3H-thymidine labelling and autoradiography, indicate that greater than 98% of the pre-confluent osteoblasts are proliferating. By two weeks, both the foci of multilayered cells and internodular cell regions have down-regulated cell growth associated genes. Post-proliferatively, but not earlier, initial expression of both osteocalcin and osteopontin are restricted to the multilayered nodules where all cells exhibit expression. While total mRNA levels for osteopontin and osteocalcin are coordinately upregulated with an increase in mineral deposition, in situ hybridization has revealed that expression of osteocalcin and osteopontin occurs predominantly in cells associated with the developing nodules. In contrast, proliferating rat osteosarcoma cells (ROS 17/2.8) concomitantly express histone H4, along with osteopontin and osteocalcin. These in situ analyses of gene expression during osteoblast growth and differentiation at the single cell level establish that a population of proliferating calvarial-derived cells subsequently expresses osteopontin and osteocalcin in cells developing into multilayered nodules with a tissue-like organization.