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PubMed2026

Decoding the landscape of cell-type-specific co-expressed transcription factors in soybean.

Soybean (Glycine max) is an essential source of protein and oil with high nutritional value for human and animal consumption. To enhance our understanding of soybean biology, it is essential to have accurate information regarding the expression of each of its protein-coding genes. Here, we present Tabula Glycine max, a soybean single-cell resolution transcriptome atlas. This atlas comprises single-nucleus RNA-sequencing data from ten different G. max organs and morphological structures constituting the entire soybean plant. These nuclei are grouped into 156 different clusters based on their transcriptomic profiles. The breadth of various organs, tissues and cell types represented in Tabula Glycine max reveals that the pattern of co-expressed transcription factor genes is sufficient to define most cell types based on their function and organ of origin. Defining cell-type-specific co-expressed transcription factor genes offers a new perspective to engineer cell-type-specific programmes and enhance the biology of unique soybean cell types. This cellular resolution and breadth make the Tabula Glycine max an exceptional resource for the plant and soybean communities.

Journal Article↗
PubMed2000

Thrombopoietin induces phosphoinositol 3-kinase activation through SHP2, Gab, and insulin receptor substrate proteins in BAF3 cells and primary murine megakaryocytes.

Thrombopoietin (TPO) is a recently characterized member of the hematopoietic growth factor family that serves as the primary regulator of megakaryocyte (MK) and platelet production. The hormone acts by binding to the Mpl receptor, the product of the cellular proto-oncogene c-mpl. Although many downstream signaling targets of TPO have been identified in cell lines, primary MKs, and platelets, the molecular mechanism(s) by which many of these molecules are activated remains uncertain. In this report we demonstrate that the TPO-induced activation of phosphoinositol 3-kinase (PI3K), a signaling intermediate vital for cellular survival and proliferation, occurs through its association with inducible signaling complexes in both BaF3 cells engineered to express Mpl (BaF3/Mpl) and in primary murine MKs. Although a direct association between PI3K and Mpl could not be demonstrated, we found that several proteins, including SHP2, Gab2, and IRS2, undergo phosphorylation and association in BaF3/Mpl cells in response to TPO stimulation, complexes that recruit and enhance the enzymatic activity of PI3K. To verify the physiological relevance of the complex, SHP2-Gab2 association was disrupted by overexpressing a dominant negative SHP2 construct. TPO-induced Akt phosphorylation was significantly decreased in transfected cells suggesting an important role of SHP2 in the complex to enhance PI3K activity. In primary murine MKs, TPO also induced phosphorylation of SHP2, its association with p85 and enhanced PI3K activity, but in contrast to the results in cell lines, neither Gab2 nor IRS2 are phosphorylated in MKs. Instead, a 100-kDa tyrosine-phosphorylated protein (pp100) co-immunoprecipitated with the regulatory subunit of PI3K. These findings support a model where PI3K activity is dependent on its recruitment into TPO-induced multiphosphoprotein complexes, implicate the existence of a scaffolding protein in primary MKs distinct from the known Gab and IRS proteins, and suggest that, in contrast to erythroid progenitor cells that employ Gab1 in PI3K signaling complexes, utilization of an alternate member of the Gab/IRS family could be responsible for specificity in TPO signaling.

Adaptor Proteins, Signal Transducing↗
PubMed2005

The cellular plasticity of human adipocytes.

Little is known regarding the biology of fat considering its extensive use clinically in soft tissue implantation. Free-fat transfer is problematic the result of graft site volume loss, appearing histologically as the replacement of mature adipocytes with a fibroblast-like infiltrate. We hypothesize that these histologic changes reflect a dedifferentiation of ischemic mature adipocytes instead of, or in addition to, a more traditional response. To explore this hypothesis, we studied the in vitro morphologic changes of cultured mature human adipocytes isolated from liposuctioned adipose tissue. Most adipocytes over time lost significant amounts of intracellular lipid. Ultimately, these cells lost all lipid, appeared fibroblastic, and proliferated to confluence. Adipogenic induction of such dedifferentiated adipocytes resulted in reaccumulation of intracellular lipid. This study demonstrates that mature adipocytes can be cultured from human liposuctioned fat, they can dedifferentiate into fibroblastic cells, and the fibroblast-like cells can be expanded and turned into lipid-synthesizing adipocytes. Exploration of this cellular plasticity might ultimately yield important insights into free-fat transfer and novel tissue-engineering strategies.

Adipocytes↗
PubMed1992

Monoclonal antibodies defining shared human macrophage-endothelial antigens.

We have selected several monoclonal antibodies (mAbs) producing using human rheumatoid arthritis (RA) synovial macrophages (m phi s) as immunogen. Of these, mAbs 8H2, 10G7 and 10G9 showed cross reactivity with endothelium, suggesting common antigens between these cell types. We have determined the spectrum of reactivity of these mAbs on hematopoietic cell lines, peripheral blood cells, and inflammatory and non-inflammatory tissues by immunohistochemistry. MAb 8H2 does not react with the myeloid cell lines HL60 (myelocytic), U937 (histiocytic lymphoma), and K562 (erythroleukemia), or with peripheral blood cells. In normal and inflamed tissue sections, mAb 8H2 reacts with m phi s and endothelial cells. In contrast, mAb 10G7 does not react with peripheral blood cells, but reacts with HL60, U937, and K562 cell lines, as well as with m phi s and endothelial cells in inflamed and noninflamed tissues. MAb 10G9 does not react with myeloid cell lines, but reacts with monocytes and platelets in peripheral blood. In both normal and inflamed tissues, mAb 10G9 reacts with m phi s and endothelial cells. The antigens identified by these three mAbs were characterized biochemically, by enzymatic digestion of RA synovial tissue m phi s followed by a cellular ELISA, as well as by reactivity of the mAbs with NIH-3T3 cells genetically engineered to express known myeloid antigens. These mAbs reacted with protein or glycoprotein antigens distinct from the known myeloid antigens CD13, CD14, CD33, CD34, CD36, and c-fms. These mAbs should prove to be a valuable tool for studying m phi s and endothelial cells and their shared antigenic determinants.

3T3 Cells↗
PubMed2005

Targeting CD20 in follicular NHL: novel anti-CD20 therapies, antibody engineering, and the use of radioimmunoconjugates.

Rituximab (chimeric anti-CD20 monoclonal antibody) is widely employed in the treatment of patients with B cell non-Hodgkin lymphoma (NHL). This agent has activity in both indolent and aggressive disease, alone and in combination with chemotherapy. Unfortunately, however, many patients develop resistant disease. Ongoing efforts to improve outcomes include changes in dose and schedule, as well as the use of other biologic agents or antibodies that may enhance activity when administered together with rituximab. A relatively new focus is the development of engineered anti-CD20 antibodies that are optimized for their capability to mediate antibody-mediated cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Human or humanized structures have also been employed to potentially improve these attributes, as well as to improve on pharmacokinetics and immunogenicity. Other studies in NHL have clearly demonstrated that radiolabeled anti-CD20 antibodies (such as I-131 tositumomab and Y-90 ibritumomab tiuxetan) may be useful in relapsed and refractory disease, and have potential utility as part of initial treatment as well. Further studies of these modified anti-CD20 antibodies are ongoing in order to optimize their use for maximal clinical benefit.

Antibodies, Monoclonal↗
PubMed1994

The coupling of multiple signal transduction pathways with steroid response mechanisms.

In a human breast carcinoma-derived cell line engineered to contain a hormone-responsive luciferase reporter gene, manipulation of cell growth conditions or cellular signal transduction in a variety of ways can enhance or impair glucocorticoid-mediated induction of a target gene. Induction may be enhanced as much as 10-fold or inhibited 90% by different treatments. For example, two different inhibitors of protein phosphatase-1 and -2A potentiated the hormone-dependent induction of luciferase. Activation of protein kinase-A via addition of 8-bromo-cAMP or forskolin also potentiated the hormonal induction, whereas 8-bromo-cGMP was ineffective. In contrast, activating protein kinase-A by inhibiting cAMP turnover with the phosphodiesterase inhibitors isobutylmethylxanthine or Ro20-1724 inhibited the hormone response rather than potentiated it. The inhibitory activity of isobutylmethylxanthine was evident even when activators of protein kinase-A are administered simultaneously. Isobutylmethylxanthine must, therefore, activate a signal transduction pathway in addition to the protein kinase-A pathway. Activation of protein kinase-C potentiated the hormone response in a cell-specific manner. Treatment with epidermal growth factor and imposition of cell stress by heat shock or inhibition of protein synthesis also enhanced the glucocorticoid response. Thus, our results suggest an elaborate coupling of the steroid response pathway with other cellular signal transduction mechanisms that permits an additional layer of control to be imposed on hormone-mediated transcriptional responses. It is proposed that cell-specific phosphorylation events influence steroid receptor interaction with the basal transcription apparatus, thereby altering receptor-mediated induction mechanisms.

Dexamethasone↗
PubMed1999

Muscle tissue engineering.

Only recently have scientists come to appreciate that many tissues and cell populations that were formerly considered to be in a terminally differentiated state are capable of division and or dedifferentiation. It is the goal of the tissue engineer to understand and redirect this potential. Muscle tissue-engineering efforts will be directed toward building in vitro replacements for in vivo problems. Tissue-engineering advances will be interdependent with advances in gene therapy techniques to restore function at a cellular level.

Animals↗
PubMed2003

A noninvasive genetic/pharmacologic strategy for visualizing cell morphology and clonal relationships in the mouse.

Analysis of cellular morphology is the most general approach to neuronal classification. With the increased use of genetically engineered mice, there is a growing need for methods that can selectively visualize the morphologies of specified subsets of neurons. This capability is needed both to define cell morphologic phenotypes and to mark cells in a noninvasive manner for lineage studies. To this end, we describe a bipartite genetic system based on a Cre-estrogen receptor (ER) fusion protein that irreversibly activates a plasma membrane-bound alkaline phosphatase reporter gene by site-specific recombination. Because the efficiency and timing of gene rearrangement is controlled pharmacologically, a sparse subset of labeled cells can be generated from the set of CreER-expressing cells at any time during development. Histochemical visualization of alkaline phosphatase activity reveals neuronal morphology with strong and uniform labeling of all processes.

Alkaline Phosphatase↗
PubMed2006

In vitro culture of human dermal fibroblasts on electrospun polycaprolactone collagen nanofibrous membrane.

Novel cost-effective electrospun nanofibrous membrane is established for wound dressing and allogeneic cultured dermal substitute through the cultivation of human dermal fibroblast for skin defects. Synthetic polymers are generally used for tissue engineering and drug delivery applications because of their remarkable mechanical stability and slow degradation. Polycaprolactone (PCL) is used as a bioresorbable polymer in numerous medical devices as well as for tissue engineering applications. The large surface area of the polymer nanofibers with specific modifications facilitates cell adhesion and control of their cellular functions. The objectives of this study was to fabricate electrospun nanofibrous membrane from biodegradable PCL for wound dressing and collagen-blended nanofibrous membrane, and to examine fibroblast attachment, cell proliferation, and morphology of cell matrix interaction. Results of the present investigation prove that the porous nanofibrous membrane is suitable for wound dressing and modified PCL-blended collagen nanofibrous membrane is suitable for the attachment and proliferation of fibroblast, and might have the potential to be applied in tissue engineering as a dermal substitute for the treatment of skin defects and burn wounds.

Biocompatible Materials↗
PubMed2006

Flow-dependent re-endothelialization of tissue-engineered heart valves.

BACKGROUND AND AIM OF THE STUDY: The generation of a functional, non-immunogenic, non-thrombogenic construct based on autologous cells seeded onto an acellular extracellular matrix is the major goal in heart valve tissue engineering. The study aim was to identify culturing conditions required to achieve a stable endothelial cell (EC) layer under physiological flow conditions, a prerequisite for the requested characteristics. METHODS: Eleven detergent-decellularized ovine pulmonary valves (PVs) were statically reseeded in special bioreactors with ovine venous ECs (1.2x10(7) cells per valve). The dynamic culture was started with 0.1 l/min in eight bioreactors. In four bioreactors the initial flow rate was slow, and increased by 0.1 l/min twice each day until maximal flow was 0.5 l/min and pulsation rate (PR) was 20 beats/min; in four other bioreactors the flow was increased by 0.7 l/min/day and reached 2.0 l/min with a PR of 50 beats/min. The mean system pressure was maintained at 25 +/- 5 mmHg during the whole dynamic cultivation in both groups. Three statically reseeded valves served as baseline. After achieving maximal appointed flow, the valves were investigated morphologically (hematoxylin and eosin staining, electron microscopy, von Willebrand factor, endothelial nitric oxide synthase immunostaining) and for metabolic activity (MTS assay). RESULTS: After reseeding, the endothelium appeared on the luminal surface of the PV as a non-confluent monolayer. Moderate pulsatile circulation induced complete confluence of EC monolayers on both cusp sides and the pulmonary wall. A high flow rate led to a partial loss of cells on the wall surface with large defects, and to complete cell wash-off from cusps. Cusp and wall metabolic activity was significantly higher after culture under moderate flow (p < 0.001) than in other groups, and was absent from cusps in high-flow bioreactors. CONCLUSION: Moderate pulsatile flow with small increments stimulates EC proliferation on the ovine decellularized valve scaffold. A rapid increase in bioreactor flow to physiological levels leads to significant damage of the reseeded endothelium and complete loss of cusp cellularity. This effect may be responsible for the in-vivo failure of static reseeded tissue-engineered valves exposed to physiological hemodynamic forces.

Animals↗
PubMed1991

Engineering T lymphocyte antigen specificity.

Targeting of immune cells by bispecific antibodies has proven to be a powerful tool for the investigation of cellular cytotoxicity, lymphocyte activation and induction of cytokine production, as well as to represent an innovative form of immunotherapy for the treatment of cancer. The hallmark of this approach is the use of the specificity of monoclonal antibodies to join target and immune cells by virtue of the dual specificity of bispecific antibodies for the two entities. More precisely, the bispecific antibody has two different binding sites, which are capable of recognizing tumor associated antigens on the one hand and lymphocyte activation sites on the other. This process of crosslinking results in the activation of the lymphocyte and triggering of its lytic machinery, as well as lymphokine production. A major advantage of this therapeutic modality is, that use is made of the normal cellular immune defence system and therefore is only associated with minor toxicity. The distinct lymphocyte populations, which can be used for adoptive immunotherapy and the various bispecific antibody preparations, as well as the chimeric immunoglobulin/T cell receptor construction, are the major topics of this review.

Animals↗
PubMed2026

Mechanotransduction in musculoskeletal mesenchymal tissues: implications for bone, tendon, and cartilage regenerative engineering-a narrative review.

PURPOSE/AIM OF THE STUDY: To integrate evidence on how mechanical signals regulate musculoskeletal connective-tissue biology and how cellular context and loading history shape mechanotransduction and mechanical memory. MATERIALS AND METHODS: This narrative review synthesized PubMed-indexed evidence on extracellular matrix mechanics, adhesion complexes, the cytoskeleton, nucleus, primary cilia, mechanosensitive ion channels, cell state, and loading history in bone, tendon, ligament, and cartilage. RESULTS: Mechanotransduction is best understood as a coupled extracellular matrix-integrin-cytoskeleton-nucleus continuum rather than as independent cytoskeletal or nuclear drivers. Responses are conditioned by lineage stage, anatomic niche, inflammation, cellular subpopulation, and prior mechanical exposure. Mechanical memory may be encoded through persistent YAP/TAZ activity, microRNA programs, DNA methylation, histone modifications, chromatin architecture, and metabolic remodeling. Evidence is strongest for bone, including Piezo-dependent osteogenesis, TRPV4-mediated shear sensing, viscoelastic compression, osteocyte-stromal extracellular-vesicle signaling, and osteogenesis-angiogenesis coupling. Tendon and ligament require anisotropic architecture and strain-window control, whereas cartilage shows a narrow distinction between physiologic TRPV4-associated anabolism and high-strain or inflammation-sensitized Piezo/YAP-mediated maladaptation. CONCLUSIONS: Translational implications include mechanically defined cell expansion, biomaterial preconditioning, stage-specific rehabilitation, and potency assays incorporating loading history. Direct clinical validation of stable perioperative cellular mechanical memory remains limited. Future studies should combine controlled mechanical perturbation with bulk and single-cell RNA sequencing, chromatin-accessibility profiling, spatial methods, and perturbational genomics.

Mechanotransduction↗
PubMed2004

Identification of cellular cofactors for human immunodeficiency virus replication via a ribozyme-based genomics approach.

Ribozymes are small, catalytic RNA molecules that can be engineered to down-regulate gene expression by cleaving specific mRNA. Here we report the selection of hairpin ribozymes that inhibit human immunodeficiency virus (HIV) replication from a combinatorial ribozyme library. We identified a total of 17 effective ribozymes, each capable of inhibiting HIV infection of human CD4(+) cells. These ribozymes target diverse steps of the viral replication cycle, ranging from entry to transcription. One ribozyme suppressed HIV integration and transcription by inhibiting the expression of the Ku80 subunit of the DNA-activated protein kinase. Another ribozyme specifically inhibited long terminal repeat transactivation, while two additional ones blocked a step that can be bypassed by vesicular stomatitis virus G-protein pseudotyping. The function of Ku80 in HIV replication and its mechanism of action were further confirmed using short interfering RNA. Identification of the gene targets of these and other selected ribozymes may reveal novel therapeutic targets for combating HIV infection.

Apoptosis↗
PubMed2001

[A study on cellular matrix via cell extraction from bovine pericardium].

OBJECTIVE: To obtain an acellular matrix from bovine pericardial tissue, as a scaffold for tissue engineering of heart valves or cardiovascular patching applications. METHODS: A four step detergent and enzyme link extraction procedure was practiced in this study. The mechanic properties of the acellular matrix were evaluated, and the components were analyzed biochemically. RESULTS: HE staining confirmed the removal of cells and Von Gieson staining showed the integrality of collagen and elastin. Biochemical analysis demonstrated the retention of collagen and some glycosaminoglycans while the percentage of the soluble proteins reduced slightly. The tissue fracture strength and shrinkage temperature made no significant difference after extraction. CONCLUSIONS: The extraction process is effective to remove cells from bovine pericardial tissue while maintains its mechanical strength. This approach may eventually lead to a scaffold for heart valves and cardiovascular patching applications in tissue engineering.

Animals↗
PubMed1999

Skeletal muscle tissue engineering using isolated myoblasts on synthetic biodegradable polymers: preliminary studies.

Skeletal muscle is responsible for the control of voluntary movement and the maintenance of structural contours of the body. Muscle loss or deficiency is encountered in various pathological states, and attempts to correct them have been employed with limited success. The aim of the present study was to tissue engineer three-dimensional vascularized skeletal muscle using isolated myoblasts attached to synthetic biodegradable polymer for tissue replacement in the enhancement of muscle regeneration. Myoblasts derived from neonatal rats (3-5-day-old), Fisher CDF-F344, were seeded onto polyglycolic acid meshes and implanted into the omentum of syngeneic adult Fisher CDF-F344 rats. Rats were sacrificed on day 30 and day 45 after the transplantation, and the cell-polymer constructs were harvested for morphological analysis. Histological analysis of the constructs were performed by hematoxylin and eosin, and immunohistochemical staining was positive for alpha sarcomeric actin and desmin skeletal muscle marker. Viable myoblasts organized between strands of degrading polymer mesh formed the new tissue, and vascularization of the entire construct was observed. Organization of neomuscle strands surrounded by vascularized tissue composed of degrading polymer and fusing myoblasts demonstrated the ability of myoblast constructs to survive, reorganize and regenerate tissue-like structures. Since myoblast transplantation to date has been limited to the cellular level of replacement, myoblast-polyglycolic acid constructs may be useful in defining the application of tissue engineering for future skeletal muscle transplantations.

Actins↗
PubMed2007

Analysis of oxygen transport in a diffusion-limited model of engineered heart tissue.

Cardiac tissue engineering has made notable progress in recent years with the advent of an experimental model based on neonatal cardiomyocytes entrapped in collage gels and purified basement membrane extract, known as "engineered heart tissues" (EHTs). EHTs are a formidable display of tissue-level contractile function and cellular-level differentiation, although they suffer greatly from mass transport limitations due to the high density of metabolically active cells and the diffusion-limited nature of the hydrogel. In this report, a mathematical model was developed to predict oxygen levels inside a one-dimensional, diffusion-limited model of EHT. These predictions were then compared to values measured in corresponding experiments with a hypoxia-sensitive stain (pimonidazole). EHTs were cast between two plastic discs, which allowed for mass transfer with the culture medium to occur in only the radial direction. EHTs were cultured for up to 36 h in the presence of pimonidazole, after which time they were snap-frozen, histologically sectioned, and stained for bound pimonidazole. Quantitative image analysis was performed to measure the distance from the culture medium at which hypoxia first occurs under various conditions. As tested by variation of simple design parameters, the trends in oxygen profiles predicted by the model are in reasonable agreement with those obtained experimentally, although a number of ambiguities related to the specific model parameters led to a general overprediction of oxygen concentrations. Based on the sensitivity analysis in the present study, it is concluded that diffusion-reaction models may offer relatively precise predictions of oxygen concentrations in diffusion-limited tissue constructs.

Biological Transport↗
PubMed2005

Controlling cell biomechanics in orthopaedic tissue engineering and repair.

Tissue engineering offers an alternative approach with great potential for the treatment or replacement of damaged tissues or organs. In contrast to current treatments, a small sample of cells can be collected from the patient and cultured in vitro, greatly increasing the number of cells available for engineering tissue implants. As a result, engineered tissue implants limit the problems associated with patient trauma and undesirable immune response currently observed in surgical treatments practised in tissue and organ replacement. Mechano-transduction is known to play an essential role in bone tissue remodelling and repair. At physiological magnitudes, the effects of secondary messenger pathways, their components and local mediators generated as a direct result of mechanical load are known to result in an elevation of specific matrix protein mRNAs. Up-regulation of matrix protein production is paramount to tissue formation. Thus, mechano-transduction offers a method of producing bone tissue in vitro. However, successful transduction of mechanical stimuli from a substrate to cells is reliant upon a number of factors including cell-substrate adhesion, scaffold material mechanics and the activation of membrane channels, for example voltage-operated calcium channels (VOCC). Our research focuses on the optimisation of mechano-transduction pathways for successful bone tissue engineering. In this paper, we focus on the effects of cell-substrate adhesion, attenuation of VOCC activation states and biological conditioning of cell-scaffold constructs utilising bioreactors in relation to mechano-transduction-induced bone tissue production. The effects of these factors on successful bone tissue formation observed in increased matrix protein synthesis due to the optimisation of mechano-transduction pathways is discussed.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗