A thin-layer model for viscoelastic, stress-relaxation testing of cells using atomic force microscopy: do cell properties reflect metastatic potential?

Atomic force microscopy has rapidly become a valuable tool for quantifying the biophysical properties of single cells. The interpretation of atomic force microscopy-based indentation tests, however, is highly dependent on the use of an appropriate theoretical model of the testing configuration. In this study, a novel, thin-layer viscoelastic model for stress relaxation was developed to quantify the mechanical properties of chondrosarcoma cells in different configurations to examine the hypothesis that viscoelastic properties reflect the metastatic potential and invasiveness of the cell using three well-characterized human chondrosarcoma cell lines (JJ012, FS090, 105KC) that show increasing chondrocytic differentiation and decreasing malignancy, respectively. Single-cell stress relaxation tests were conducted at 2 h and 2 days after plating to determine cell mechanical properties in either spherical or spread morphologies and analyzed using the new theoretical model. At both time points, JJ012 cells had the lowest moduli of the cell lines examined, whereas FS090 typically had the highest. At 2 days, all cells showed an increase in stiffness and a decrease in apparent viscosity compared to the 2-h time point. Fluorescent labeling showed that the F-actin structure in spread cells was significantly different between FS090 cells and JJ012/105KC cells. Taken together with results of previous studies, these findings indicate that cell transformation and tumorigenicity are associated with a decrease in cell modulus and apparent viscosity, suggesting that cell mechanical properties may provide insight into the metastatic potential and invasiveness of a cell.     

Up-regulation of matrix metalloproteinase-9 in primary bone tumors and its association with tumor aggressiveness

Molecular Biology Reports - Understanding the molecular mechanism underlying the pathophysiology of primary skeletal tumors is crucial due to the tumor-related complications, incidence at...     

Construction and functional characterization of a fully human anti-CD19 chimeric antigen receptor (huCAR)-expressing primary human T cells

Although remarkable results have been attained by adoptively transferring T cells expressing fully murine and/or humanized anti-CD19 chimeric antigen receptors (CARs) to treat B cell malignancies, evidence of human anti-mouse immune responses against CARs provides a rationale for the development of less immunogenic CARs. By developing a fully human CAR (huCAR), these human anti-mouse immune responses are likely eliminated. This, perhaps, not only increases the persistence of anti-CD19 CAR T cells—thereby reducing the risk of tumor relapse—but also facilitates administration of multiple, temporally separated doses of CAR T cells to the same recipient. To these ends, we have designed and constructed a second-generation fully human anti-CD19 CAR (or huCAR19) containing a fully human single-chain variable fragment (ScFv) fused with a CD8a hinge, a 4-1BB transmembrane domain and intracellular T cell signaling domains of 4-1BB and CD3z. T cells expressing this CAR specifically recognized and lysed CD19⁺ target cells produced cytokines and proliferated in vitro. Moreover, cell volume data revealed that our huCAR construct cannot induce antigen-independent tonic signaling in the absence of cognate antigen. Considering our results, our anti-CD19 huCAR may overcome issues of transgene immunogenicity that plague trials utilizing CARs containing mouse-derived ScFvs. These results suggest that this huCAR19 be safely and effectively applied for adaptive T cell immunotherapy in clinical practice.     

Effect of resveratrol on the radiosensitivity of 5-FU in human breast cancer MCF-7 cells

The aim of this study was to assess the efficacy of resveratrol (Res) on radiosensitivity of 5-fluorouracil (5-FU) in the spheroid culture of MCF-7 breast cancer cell line using colony formation examination. Spheroids on day 9 with 300 µm diameters were treated with 20 µM resveratrol and/or 1 µM 5-FU for one volume doubling time (VDT) (42 hours) and then irradiated with 2 Gy gamma radiation (⁶⁰Co) in various groups. Then the viability of the cells and clonogenic ability were acquired by blue dye exclusion and colony formation assay, respectively. The population doubling time in the monolayer culture and the VDT of spheroid culture was 22.48 0.23 hours and 42 0.63 hours respectively. None of the drugs and combination of them had any effect on the viability of cells. The combination treatment of 5-FU+Res+ radiation significantly reduced the colony formation ability of spheroid cells in comparison with each treatment alone. Our results indicated that resveratrol can significantly decrease colony number of breast cancer spheroid cells treated with 5-FU in combination with gamma-rays. Thus, resveratrol as a hypoxia-inducible factor-1-alpha inhibitor increased the radiosensitization of breast cancer spheroid cells.     

Plant community responses to environmentally friendly piste management in northeast Iran

It is well‐known that pistes have adverse effects on alpine ecosystems. Previous studies urged that pistes should be installed and managed in the ways to minimize negative impacts on natural habitats. However, the impacts of this type of management on the plant communities are not widely studied. The aim of this study was to examine species composition and biodiversity changes in an environmentally friendly managed piste in northeast Iran. This piste has been established in a previously degraded alpine landscape. For the vegetation survey, we sampled 44 within and 28 off‐piste plots. Except for the piste management, other environmental factors were similar between the piste and off‐piste plots. Dominant species were determined, and variation in community composition of the two areas was visualized. Also, native species, phylogenetic, and functional Hill diversity of the two areas were compared. The results showed that there was a moderate differentiation in the species composition of the piste and off‐piste. Two palatable species (i.e., Bupleurum falcatum and Melica persica) were dominant in the piste and were not recorded in the off‐piste. The diversity calculations results showed that the species diversity of the piste was higher than that of the off‐piste. Phylogenetic diversity at the level of frequent and dominant plants showed a similar result. The piste had a higher functional diversity in terms of functional richness, and functional diversity of frequent and dominant plants. Our findings imply, after 10 years, species, phylogenetic, and functional diversity of the piste is significantly improved. Environmentally friendly piste management (EFPM) induced species composition change that led to emerging species that were absent in the off‐piste. We can conclude that EFPM led to restoration of a degraded landscape. Long‐term impacts of EFPM are still unknown, therefore, caution should be undertaken regarding the installation of new environmentally friendly pistes in other areas.     

Effects of livestock grazing on soil,plant functional diversity,and ecological traits vary between regions with different climates in northeastern Iran

Understanding the responses of vegetation characteristics and soil properties to grazing in different precipitation regimes is useful for the management of rangelands, especially in the arid regions. In northeastern Iran, we studied the responses of vegetation to livestock grazing in three regions with different climates: arid, semiarid, and subhumid. In each region, we selected 6–7 pairwise sampling areas of high versus low grazing intensity and six traits of the present species were recorded on 1 m² plots—five grazed and five ungrazed in each area. The overall fertility was compared using the dissimilarity analysis, and linear mixed‐effect models were used to compare the individual fertility parameters, functional diversity indices, and species traits between the plots with high and low grazing intensity and between the climatic regions. Both climate and grazing, as well as their interaction, affected fertility parameters, functional diversity indices, and the representation of species traits. Grazing reduced functional evenness, height of the community, the representation of annuals, but increased the community leaf area. In the subhumid region, grazing also reduced functional richness. Further, grazing decreased the share of annual species in the semiarid region and seed mass in the arid region. Larger leaf area and seed mass, smaller height and lower share of annuals were associated with intensive grazing. Species with large LA and seed mass, lower height and perennials can be therefore presumed to tolerate trampling and benefit from high nutrient levels, associated with intensive grazing. By providing a detailed view on the impacts of overgrazing, this study highlights the importance of protection from grazing as an effective management tool for maintaining the pastoral ecosystems. In general, the composition of plant traits across the pastures of northeastern Iran was more affected by intensive grazing than by the differences in climate.     

Design, synthesis, and anticancer activity of phosphonic acid diphosphate derivative of adenine-containing butenolide and its water-soluble derivatives of paclitaxel with high antitumor activity

Synthesis of adenine derivative of triphosphono-gamma-(Z)-ethylidene-2,3-dimethoxybutenolide 4 was accomplished by treatment of phosphonate 3 with 5-phosphoribosyl 1-pyrophosphate in the presence of 5-phosphoribosyl 1-pyrophosphate synthetase. It was found that triphosphonate 4 functions as an irreversible stoichiometric inactivator of the Escherichia coli ribonucleoside diphosphate reductase (RDPR). Triphosphonate 4 exhibited potent inhibitory activity against murine leukemias (L1210 and P388), breast carcinoma (MCF7), and human T-lymphoblasts (Molt4/C8 and CEM/0) cell lines. Paclitaxel ester derivatives of adenine-containing triphosphono-gamma-(Z)-ethylidene-2,3-dimethoxybutenolide 8-10 were also synthesized. Like triphosphonate 4, compound 8 exhibited inhibitory property toward RDPR. It also induced microtubule assembly similar to paclitaxel (5). The structure of the chlorodiester linker in 8 was found to account for this dual property. After treatment of MCF7 cells with compounds 4, 5, and 8, fluorescence microscope examination demonstrated the presence of nucleus shrinkage or segmentation. Bifunctional prodrug 8 exhibited higher lipophilicity than 4 and higher water-solubility than 5. Pro-dual-drug 8 exhibited more pronounced anticancer activity relative to that of the triphosphonate 4 and paclitaxel (5). In contrast, compound 9, resulting from the linkage of triphosphonate 4 and paclitaxel (5) through a diester unit, was only found to function as a highly water-soluble prodrug for paclitaxel (5). It induced microtubule assembly in vitro, but did not show inhibitory property toward RDPR. On the other hand, compound 10, an aggregate of triphosphonate 4 and paclitaxel (5), neither functioned as an inhibitor of RDPR nor exhibited microtubule assembly stimulating activity in vitro.     

Integrative repair of the meniscus: lessons from in vitro studies

Current therapies for meniscal injury seek to preserve and repair damaged tissue since loss of meniscal tissue is associated with degenerative changes in the joint, ultimately leading to osteoarthritis (OA). After a meniscal tear, the difficulty of integrating juxtaposed meniscal surfaces continues to be an obstacle. In order to determine the local factors that are necessary for successful tissue repair, previous studies have developed in vitro model systems that allow both biological and quantitative biomechanical measures of meniscus repair. Many studies have shown the importance of individual factors in meniscus metabolism, but there is a complex interplay among a variety of factors that influence meniscal healing, including inflammatory cytokines, growth factors, mechanical loading, and zonal differences in cell and tissue properties. In particular, the upregulation of inflammatory cytokines following joint injury appears to have significant catabolic influences on meniscal cell metabolic activity that must be overcome in order to promote repair. In the presence of inflammatory cytokines, such as interleukin-1 (IL-1) or tumor necrosis factor alpha (TNF-alpha), intrinsic meniscal repair in vitro is significantly inhibited. While anabolic growth factors, such as transforming growth factor-beta1 (TGF-beta1), enhance meniscal repair, they cannot completely overcome the IL-1-mediated inhibition of repair. The mechanisms by which these mediators influence meniscal repair, and their interactions with other factors in the microenvironment, such as mechanical loading, remain to be determined. Future studies must address these complex interactions during meniscal healing to ultimately enhance meniscal repair.     

Viscoelastic properties of human mesenchymally-derived stem cells and primary osteoblasts, chondrocytes, and adipocytes

The mechanical properties of single cells play important roles in regulating cell-matrix interactions, potentially influencing the process of mechanotransduction. Recent studies also suggest that cellular mechanical properties may provide novel biological markers, or "biomarkers," of cell phenotype, reflecting specific changes that occur with disease, differentiation, or cellular transformation. Of particular interest in recent years has been the identification of such biomarkers that can be used to determine specific phenotypic characteristics of stem cells that separate them from primary, differentiated cells. The goal of this study was to determine the elastic and viscoelastic properties of three primary cell types of mesenchymal lineage (chondrocytes, osteoblasts, and adipocytes) and to test the hypothesis that primary differentiated cells exhibit distinct mechanical properties compared to adult stem cells (adipose-derived or bone marrow-derived mesenchymal stem cells). In an adherent, spread configuration, chondrocytes, osteoblasts, and adipocytes all exhibited significantly different mechanical properties, with osteoblasts being stiffer than chondrocytes and both being stiffer than adipocytes. Adipose-derived and mesenchymal stem cells exhibited similar properties to each other, but were mechanically distinct from primary cells, particularly when comparing a ratio of elastic to relaxed moduli. These findings will help more accurately model the cellular mechanical environment in mesenchymal tissues, which could assist in describing injury thresholds and disease progression or even determining the influence of mechanical loading for tissue engineering efforts. Furthermore, the identification of mechanical properties distinct to stem cells could result in more successful sorting procedures to enrich multipotent progenitor cell populations.