A particle-based micromechanics approach to simulate structural changes of plant cells during drying

This paper is concerned with applying a particle-based approach to simulate the micro-level cellular structural changes of plant cells during drying. The objective of the investigation was to relate the micro-level structural properties such as cell area, diameter and perimeter to the change of moisture content of the cell. Model assumes a simplified cell which consists of two basic components, cell wall and cell fluid. The cell fluid is assumed to be a Newtonian fluid with higher viscosity compared to water and cell wall is assumed to be a visco-elastic solid boundary located around the cell fluid. Cell fluid is modelled with Smoothed Particle Hydrodynamics (SPH) technique and for the cell wall; a Discrete Element Method (DEM) is used. The developed model is two-dimensional, but accounts for three-dimensional physical properties of real plant cells. Drying phenomena is simulated as fluid mass reductions and the model is used to predict the above mentioned structural properties as a function of cell fluid mass. Model predictions are found to be in fairly good agreement with experimental data in literature and the particle-based approach is demonstrated to be suitable for numerical studies of drying related structural deformations. Also a sensitivity analysis is included to demonstrate the influence of key model parameters to model predictions.

Closed system isolation and scalable expansion of human placental mesenchymal stem cells

Mesenchymal stem cells (MSC) are emerging as a leading cellular therapy for a number of diseases. However, for such treatments to become available as a routine therapeutic option, efficient and cost-effective means for industrial manufacture of MSC are required. At present, clinical grade MSC are manufactured through a process of manual cell culture in specialized cGMP facilities. This process is open, extremely labor intensive, costly, and impractical for anything more than a small number of patients. While it has been shown that MSC can be cultivated in stirred bioreactor systems using microcarriers, providing a route to process scale-up, the degree of numerical expansion achieved has generally been limited. Furthermore, little attention has been given to the issue of primary cell isolation from complex tissues such as placenta. In this article we describe the initial development of a closed process for bulk isolation of MSC from human placenta, and subsequent cultivation on microcarriers in scalable single-use bioreactor systems. Based on our initial data, we estimate that a single placenta may be sufficient to produce over 7,000 doses of therapeutic MSC using a large-scale process.

Neural differentiation of mouse embryonic stem cells on conductive nanofiber scaffolds

Nerve tissue engineering requires suitable precursor cells as well as the necessary biochemical and physical cues to guide neurite extension and tissue development. An ideal scaffold for neural regeneration would be both fibrous and electrically conductive. We have contrasted the growth and neural differentiation of mouse embryonic stem cells on three different aligned nanofiber scaffolds composed of poly L: -lactic acid supplemented with either single- or multi-walled carbon-nanotubes. The addition of the nanotubes conferred conductivity to the nanofibers and promoted mESC neural differentiation as evidenced by an increased mature neuronal markers expression. We propose that the conductive scaffold could be a useful tool for the generation of neural tissue mimics in vitro and potentially as a scaffold for the repair of neural defects in vivo.

Micromarrows—Three-dimensional coculture of hematopoietic stem cells and mesenchymal stromal cells

Hematopoietic stem cell (HSC) transplant is a well established curative therapy for some hematological malignancies. However, achieving adequate supply of HSC from some donor tissues can limit both its application and ultimate efficacy. The theory that this limitation could be overcome by expanding the HSC population before transplantation has motivated numerous laboratories to develop ex vivo expansion processes. Pioneering work in this field utilized stromal cells as support cells in cocultures with HSC to mimic the HSC niche. We hypothesized that through translation of this classic coculture system to a three-dimensional (3D) structure we could better replicate the niche environment and in turn enhance HSC expansion. Herein we describe a novel high-throughput 3D coculture system where murine-derived HSC can be cocultured with mesenchymal stem/stromal cells (MSC) in 3D microaggregates—which we term “micromarrows.” Micromarrows were formed using surface modified microwells and their ability to support HSC expansion was compared to classic two-dimensional (2D) cocultures. While both 2D and 3D systems provide only a modest total cell expansion in the minimally supplemented medium, the micromarrow system supported the expansion of approximately twice as many HSC candidates as the 2D controls. Histology revealed that at day 7, the majority of bound hematopoietic cells reside in the outer layers of the aggregate. Quantitative polymerase chain reaction demonstrates that MSC maintained in 3D aggregates express significantly higher levels of key hematopoietic niche factors relative to their 2D equivalents. Thus, we propose that the micromarrow platform represents a promising first step toward a high-throughput HSC 3D coculture system that may enable in vitro HSC niche recapitulation and subsequent extensive in vitro HSC self-renewal.

Gene expression profile of primary prostate epithelial and stromal cells in response to sulforaphane or iberin exposure

BACKGROUND: Broccoli consumption has been associated with a reduced risk of prostate cancer. Isothiocyanates (ITCs) derived from glucosinolates that accumulate in broccoli are dietary compounds that may mediate these health effects. Sulforaphane (SF, 4-methylsulphinylbutyl ITC) derives from heading broccoli (calabrese) and iberin (IB, 3-methylsulphinypropyl ITC) from sprouting broccoli. While there are many studies regarding the biological activity of SF, mainly undertaken with cancerous cells, there are few studies associated with IB. METHODS: Primary epithelial and stromal cells were derived from benign prostatic hyperplasia tissue. Affymetrix U133 Plus 2.0 whole genome arrays were used to compare global gene expression between these cells, and to quantify changes in gene expression following exposure to physiologically appropriate concentrations of SF and IB. Ontology and pathway analyses were used to interpret results. Changes in expression of a subset of genes were confirmed by real-time RT-PCR. RESULTS: Global gene expression profiling identified epithelial and stromal-specific gene expression profiles. SF induced more changes in epithelial cells, whereas IB was more effective in stromal cells. Although IB and SF induced different changes in gene expression in both epithelial and stromal cells, these were associated with similar pathways, such as cell cycle and detoxification. Both ITCs increased expression of PLAGL1, a tumor suppressor gene, in stromal cells and suppressed expression of the putative tumor promoting genes IFITM1, CSPG2, and VIM in epithelial cells. CONCLUSION: These data suggest that IB and SF both alter genes associated with cancer prevention, and IB should be investigated further as a potential chemopreventative agent.

INT6/EIF3E interacts with ATM and is required for proper execution of the DNA damage response in human cells

Altered expression of the INT6 gene, encoding the e subunit of the translational initiation factor eIF3, occurs in human breast cancers, but how INT6 relates to carcinogenesis remains unestablished. Here, we show that INT6 is involved in the DNA damage response. INT6 was required for cell survival following γ-irradiation and G(2)-M checkpoint control. RNA interference-mediated silencing of INT6 reduced phosphorylation of the checkpoint kinases CHK1 and CHK2 after DNA damage. In addition, INT6 silencing prevented sustained accumulation of ataxia telangiectasia mutated (ATM) at DNA damage sites in cells treated with γ-radiation or the radiomimetic drug neocarzinostatin. Mechanistically, this result could be explained by interaction of INT6 with ATM, which together with INT6 was recruited to the sites of DNA damage. Finally, INT6 silencing also reduced ubiquitylation events that promote retention of repair proteins at DNA lesions. Accordingly, accumulation of the repair factor BRCA1 was defective in the absence of INT6. Our findings reveal unexpected and striking connections of INT6 with ATM and BRCA1 and suggest that the protective action of INT6 in the onset of breast cancers relies on its involvement in the DNA damage response.

Contribution of DEAF1 structural domains to the interaction with the breast cancer oncogene LMO4

The proteins LMO4 and DEAF1 contribute to the proliferation of mammary epithelial cells. During breast cancer LMO4 is upregulated, affecting its interaction with other protein partners. This may set cells on a path to tumour formation. LMO4 and DEAF1 interact, but it is unknown how they cooperate to regulate cell proliferation. In this study, we identify a specific LMO4-binding domain in DEAF1. This domain contains an unstructured region that directly contacts LMO4, and a coiled coil that contains the DEAF1 nuclear export signal (NES). The coiled coil region can form tetramers and has the typical properties of a coiled coil domain. Using a simple cell-based assay, we show that LMO4 modulates the activity of the DEAF NES, causing nuclear accumulation of a construct containing the LMO4-interaction region of DEAF1.

The cancer stem-cell hypothesis : its emerging role in lung cancer biology and its relevance for future therapy

The cancer stem-cell (CSC) hypothesis suggests that there is a small subset of cancer cells that are responsible for tumor initiation and growth, possessing properties such as indefinite self-renewal, slow replication, intrinsic resistance to chemotherapy and radiotherapy, and an ability to give rise to differentiated progeny. Through the use of xenotransplantation assays, putative CSCs have been identified in many cancers, often identified by markers usually expressed in normal stem cells. This is also the case in lung cancer, and the accumulated data on side population cells, CD133, CD166, CD44 and ALDH1 are beginning to clarify the true phenotype of the lung cancer stem cell. Furthermore, it is now clear that many of the pathways of normal stem cells, which guide cellular proliferation, differentiation, and apoptosis are also prominent in CSCs; the Hedgehog (Hh), Notch, and Wnt signaling pathways being notable examples. The CSC hypothesis suggests that there is a small reservoir of cells within the tumor, which are resistant to many standard therapies, and can give rise to new tumors in the form of metastases or relapses after apparent tumor regression. Therapeutic interventions that target CSC pathways are still in their infancy and clinical data of their efficacy remain limited. However Smoothened inhibitors, gamma-secretase inhibitors, anti-DLL4 antagonists, Wnt antagonists, and CBP/β-catenin inhibitors have all shown promising anticancer effects in early studies. The evidence to support the emerging picture of a lung cancer CSC phenotype and the development of novel therapeutic strategies to target CSCs are described in this review.

Nagelschmidtite bioceramics with osteostimulation properties : material chemistry activating osteogenic genes and WNT signalling pathway of human bone marrow stromal cells

Bioactive materials with osteostimulation properties are of great importance to promote osteogenic differentiation of human bone marrow stromal cells (hBMSCs) for potential bone regeneration. We have recently synthesized nagelschmidtite (NAGEL, Ca7Si2P2O16) ceramic powders which showed excellent apatite-mineralization ability. The aim of this study was to investigate the interaction of hBMSCs with NAGEL bioceramic bulks and their ionic extracts, and to explore the osteostimulation properties of NAGEL bioceramics and the possible molecular mechanism. The cell attachment, proliferation, bone-related gene expression (ALP, OPN and OCN) and WNT signalling pathways (WNT3a, FZD6, AXIN2 and CTNNB) of hBMSCs cultured on NAGEL bioceramic disks were systematically studied. We further investigated the biological effects of ionic products from NAGEL powders on cell proliferation and osteogenic differentiation of hBMSCs by culturing cells with NAGEL extracts. Furthermore, the effect of NAGEL bioceramics on the osteogenic differentiation in hBMSCs was also investigated with the addition of cardamonin, a WNT inhibitor. The results showed that NAGEL bioceramic disks supported the attachment and proliferation of hBMSCs, and significantly enhanced the bone-related gene expression and WNT signalling pathway of hBMSCs, compared to conventional beta-tricalcium phosphate (β-TCP) bioceramic disks and blank controls. The ionic products from NAGEL powders also significantly promoted the proliferation, bone and WNT-related gene expression of hBMSCs. It was also identified that NAGEL bioceramics could bypass the action of the WNT inhibitor (10 μM) to stimulate the selected osteogenic genes in hBMSCs. Our results suggest that NAGEL bioceramics possess excellent in vitro osteostimulation properties. The possible mechanism for the osteostimulation may be directly related to the released Si, Ca and P-containing ionic products from NAGEL bioceramics which activate bone-related gene expression and WNT signalling pathway of hBMSCs. The present study suggests that NAGEL bioceramics are a potential bone regeneration material with significant osteostimulation capacity.

Cytokine synthesis by intestinal intraepithelial lymphocytes. Both gamma/delta T cell receptor-positive and alpha/beta T cell receptor-positive T cells in the G1 phase of cell cycle produce IFN-gamma and IL-5

Murine intestinal intraepithelial lymphocytes (IEL) have been shown to contain subsets of alpha/beta TCR+ and gamma/delta TCR+ T cells that spontaneously produce cytokines such as IFN-gamma and IL-5. We have now determined the nature and cell cycle stage of these cytokine-producing T lymphocytes in EIL by using IFN-gamma- and IL-5-specific ELISPOT assay, cytokine-specific mRNA-cDNA dot-blot hybridization and polymerase chain reaction, and flow cytometry (FACS) for DNA analysis. When CD3+ T cells from IEL of normal C3H/HeN mice were separated into low and high density fractions by discontinuous Percoll gradients, IFN-gamma and IL-5 spot-forming cells were only found in the former population. Analysis of mRNA for these cytokines by both IFN-gamma- and IL-5-specific dot-blot hybridization and polymerase chain reaction revealed that higher levels of message for IFN-gamma and IL-5 were also seen in the low density fraction. However, cell cycle analysis of these two fractions by FACS using propidium iodide showed a similar pattern of cell cycle stages in both low and high density populations (G0 + G1 approximately 96 to 98% and S/G2 + M approximately 2 to 4%). Finally, mRNA from gamma/delta TCR+ and alpha/beta TCR+ T cells in both low and high density fractions of IEL were analyzed for IFN-gamma and IL-5 message by polymerase chain reaction. After 35 cycles of amplification, both gamma/delta TCR+ and alpha/beta TCR+ T cells in the low density fraction expressed higher levels of message for these two cytokines when compared with the high density population. These results have now shown that both gamma/delta and alpha/beta TCR+ IEL can be separated into low and high density subsets and both fractions possess a similar stage of cell cycle. However, only the low density cells (in G1 phase) of both gamma/delta and alpha/beta TCR types possess increased cytokine-specific mRNA and produce the cytokines IFN-gamma and IL-5. Our results suggest that alpha/beta TCR+ and gamma/delta TCR+ IEL can produce cytokines without cell proliferation.

Effect of bone morphogenetic protein-4 (BMP-4) on the expression of Sox2, Oct-4 and c-Myc in human periodontal ligament cells during long-term culture

Recent studies demonstrated endogenous expression level of Sox2, Oct-4 and c-Myc is correlated with the pluripotency and successful induction of induced pluripotent stem cells (iPSCs). Periondontal ligament cells (PDLCs)have multi-lineage diferentiation capability and ability to maintain undifferentiated stage, which makes PDLCs a suitable cell source for tissue repair and regeneration. To elucidate the effect of in vitro culture condition on the stemness potential of PDLCs, we explored the cell growth, proliferation, cell cycle, and the expression of Sox2, Oct-4 and c-Myc in PDLCs from passage 1 to 7 with or without the addition of recombinant human BMP4(rhBMP4). Our results revealed that BMP-4 promoted cell growth and proliferation, arrested PDLCs in S phase of cell cycle and upregulated PI value. It was revealed that without the addition of rhBMP4, the expression of Sox2, Oct-4 and c-Myc in PDLCs only maintained nucleus location until passage 3, then lost nucleus location subsequently. The mRNA expression in PDLCs further confirmed that the level of Sox2 and Oct-4 peaked at passage 3, then decreased afterwards, whereas c-Myc maintained consistently upregulation along passages. after the treatment with rhBMP4, the expression of Sox2, Oct-4 and c-Myc in PDLCs maintained nucleus location even at passage 7 and the mRNA expression of Sox2 and Oct-4 significantly upregulated at passage 5 and 7. These results demonstrated that addition of rhBMP-4 in the culture media could improve the current culture condition for PDLCs to maintain in an undifferentiated stage.

Selective cleavage of human sex hormone-binding globulin by kallikrein-related peptidases and effects on androgen action in LNCaP prostate cancer cells

Stimulation of the androgen receptor via bioavailable androgens, including testosterone and testosterone metabolites, is a key driver of prostate development and the early stages of prostate cancer. Androgens are hydrophobic and as such require carrier proteins, including sex hormone-binding globulin (SHBG), to enable efficient distribution from sites of biosynthesis to target tissues. The similarly hydrophobic corticosteroids also require a carrier protein whose affinity for steroid is modulated by proteolysis. However, proteolytic mechanisms regulating the SHBG/androgen complex have not been reported. Here, we show that the cancer-associated serine proteases, kallikrein-related peptidase (KLK)4 and KLK14, bind strongly to SHBG in glutathione S-transferase interaction analyses. Further, we demonstrate that active KLK4 and KLK14 cleave human SHBG at unique sites and in an androgen-dependent manner. KLK4 separated androgen-free SHBG into its two laminin G-like (LG) domains that were subsequently proteolytically stable even after prolonged digestion, whereas a catalytically equivalent amount of KLK14 reduced SHBG to small peptide fragments over the same period. Conversely, proteolysis of 5α-dihydrotestosterone (DHT)-bound SHBG was similar for both KLKs and left the steroid binding LG4 domain intact. Characterization of this proteolysis fragment by [(3)H]-labeled DHT binding assays revealed that it retained identical affinity for androgen compared with full-length SHBG (dissociation constant = 1.92 nM). Consistent with this, both full-length SHBG and SHBG-LG4 significantly increased DHT-mediated transcriptional activity of the androgen receptor compared with DHT delivered without carrier protein. Collectively, these data provide the first evidence that SHBG is a target for proteolysis and demonstrate that a stable fragment derived from proteolysis of steroid-bound SHBG retains binding function in vitro.