Inactive Cells Research Articles

Pim-3 promotes human pancreatic cancer growth by regulating tumor vasculogenesis.

Pim-3, a proto-oncogene with serine/threonine kinase activity, is aberrantly expressed in malignant lesions, but not in normal pancreatic tissues. To assess the role of Pim-3 in human pancreatic carcinogenesis invivo and to determine the underlying Pim-3 signaling regulatory mechanisms, we established MiaPaca-2 cells overexpressing wild-type Pim-3 or Pim-3 kinase dead mutants (K69M-Pim-3) as well as PCI55 cells stably expressing Pim-3 shRNA or scrambled shRNA in a tetracycline-inducible manner. In addition, we conducted studies utilizing a nude mouse tumor xenograft model. Our results demonstrated that cells stably overexpressing wild-type Pim-3 exhibited functionally enhanced phosphorylation of Bad at Ser112 and increased proliferation. In contrast, the stable inactivation of Pim-3 by K69M-Pim-3 or silencing of Pim-3 expression by Pim-3 shRNA resulted in functionally decreased phosphorylation of Bad at Ser112 and higher apoptotic cells. Following subcutaneous injection of these stable cell lines, nude mice injected with Pim-3 overexpressing cells developed 100% subcutaneous tumors, together with increased PCNA-positive cells and enhanced intratumoral CD31-positive vascular areas. On the other hand, intratumoral neovascularization and tumor cell proliferation was attenuated in mice injected with Pim-3 kinase inactive cells, eventually reducing tumorigenicity in these mice to 46.6%. Moreover, Pim-3 overexpression upregulated the intratumoral levels of pSTAT3Try705, pSurvivinThr34, HGF, EGF, FGF-2 and VEGF, while the increases were markedly diminished on Pim-3 kinase inactivation. Collectively, the Pim-3 kinase emerges as being involved in accelerating human pancreatic cancer development and in promoting tumor neovascularization and subsequent tumor growth. Targeting Pim-3 may play a dual role in halting tumor progression, by promoting tumor cell death and blocking angiogenesis.

A quiescent path to plant longevity

The giant sequoia and the bristlecone pine trees are capable of living up to several hundreds or even thousands of years. Plants achieve this longevity by regenerating stem cells capable of giving rise to all differentiated cells. Plant stem cells reside in specific niches with high mitotic activity that are known as meristems. Remarkably, at the center of the root stem cell niche (SCN) resides a group of mitotically inactive cells known as the quiescent center (QC). Recent studies suggest that stress-related phytohormones and DNA damage can initiate QC cell division, resulting in the replenishment of stem cells surrounding the QC. We therefore propose that the QC represents a pool of backup cells that serve to replace damaged stem cells, thereby contributing to plant longevity.

Kinase-Independent Mechanisms of Resistance of Leukemia Stem Cells to Tyrosine Kinase Inhibitors

Tyrosine kinase inhibitors such as imatinib mesylate have changed the clinical course of chronic myeloid leukemia; however, the observation that these inhibitors do not target the leukemia stem cell implies that patients need to maintain lifelong therapy. The mechanism of this phenomenon is unclear: the question of whether tyrosine kinase inhibitors are inactive inside leukemia stem cells or whether leukemia stem cells do not require breakpoint cluster region (Bcr)-Abl signaling is currently under debate. Herein, I propose an alternative model: perhaps the leukemia stem cell requires Bcr-Abl, but is dependent on its kinase-independent functions. Kinases such as epidermal growth factor receptor and Janus kinase 2 possess kinase-independent roles in regulation of gene expression; it is worth investigating whether Bcr-Abl has similar functions. Mechanistically, Bcr-Abl is able to activate the Ras, phosphatidylinositol 3-kinase/Akt, and/or the Src-kinase Hck/Stat5 pathways in a scaffolding-dependent manner. Whereas the scaffolding activity of Bcr-Abl with Grb2 is dependent on autophosphorylation, kinases such as Hck can use Bcr-Abl as substrate, inducing phosphorylation of Y177 to enable scaffolding ability in the absence of Bcr-Abl catalytic activity. It is worth investigating whether leukemia stem cells exclusively express kinases that are able to use Bcr-Abl as substrate. A kinase-independent role for Bcr-Abl in leukemia stem cells would imply that drugs that target Bcr-Abl's scaffolding ability or its DNA-binding ability should be used in conjunction with current therapeutic regimens to increase their efficacy and eradicate the stem cells of chronic myeloid leukemia.

Role of Active and Inactive Cytotoxic Immune Response in Human Immunodeficiency Virus Dynamics

ObjectivesMathematical models can be helpful to understand the complex dynamics of human immunodeficiency virus infection within a host. Most of work has studied the interactions of host responses and virus in the presence of active cytotoxic immune cells, which decay to zero when there is no virus. However, recent research highlights that cytotoxic immune cells can be inactive but never be depleted. MethodsWe propose a mathematical model to investigate the human immunodeficiency virus dynamics in the presence of both active and inactive cytotoxic immune cells within a host. We explore the impact of the immune responses on the dynamics of human immunodeficiency virus infection under different disease stages. ResultsStandard mathematical and numerical analyses are presented for this new model. Specifically, the basic reproduction number is computed and local and global stability analyses are discussed. ConclusionOur results can give helpful insights when designing more effective drug schedules in the presence of active and inactive immune responses.

Artificial Klebsiella pneumoniae biofilm model mimicking in vivo system: altered morphological characteristics and antibiotic resistance

The purpose of this study was to develop a biofilm model of Klebsiella pneumoniae B5055, mimicking in vivo biofilm system so as to determine susceptibility of different phases of biofilm to antibiotics by three-dimensional analysis. Artificial mature biofilm of K. pneumoniae was made on black, polycarbonate membranes. Biofilm structure was visualized by scanning electron microscope (SEM) and confocal laser scanning microscopy (CLSM). Viable count method, CLSM and SEM analysis confirmed that mature, uniform and viable biofilms can be formed on the polycarbonate membranes by this method. The three-dimensional heterogeneity of biofilm was confirmed on the basis of results of CLSM, which is an important characteristics of in vivo biofilm system. Staining with the LIVE/DEAD BacLight viability kit and acridine orange suggested that the center of biofilm had more inactive cells compared with actively dividing cells on the periphery. Amikacin at a concentration of 40 μg ml⁻¹ was effective against younger biofilm whereas ineffective against older biofilm that showed sparsely populated dead cells using the BacLight viability staining kit. Role of altered morphological characteristics toward increased antibiotic susceptibility was also studied for different phases of K. pneumoniae biofilm by CLSM and light microscopy. Thickness of biofilm increased from 0.093 to 0.231 mm with time. So, both heterogeneity and thickness of the biofilm are likely to influence the ineffectiveness of amikacin in older biofilm. The present model holds considerable clinical relevance and may be useful for evaluating the efficacy of antimicrobial agent on bacterial biofilms in vitro.

Stochastic simulation of prokaryotic two-component signalling indicates stochasticity-induced active-state locking and growth-rate dependent bistability.

Signal transduction by prokaryotes almost exclusively relies on two-component systems for sensing and responding to (extracellular) signals. Here, we use stochastic models of two-component systems to better understand the impact of stochasticity on the fidelity and robustness of signal transmission, the outcome of autoregulatory gene expression and the influence of cell growth and division. We report that two-component systems are remarkably robust against copy number fluctuations of the signalling proteins they are composed of, which enhances signal transmission fidelity. Furthermore, we find that due to stochasticity these systems can get locked in an active state for extended time periods when (initially high) signal levels drop to zero. This behaviour can contribute to a bet-hedging adaptation strategy, aiding survival in fluctuating environments. Additionally, autoregulatory gene expression can cause two-component systems to become bistable at realistic parameter values. As a result, two sub-populations of cells can co-exist-active and inactive cells, which contributes to fitness in unpredictable environments. Bistability proved robust with respect to cell growth and division, and is tunable by the growth rate. In conclusion, our results indicate how single cells can cope with the inevitable stochasticity occurring in the activity of their two-component systems. They are robust to disadvantageous fluctuations that scramble signal transduction and they exploit beneficial stochasticity that generates fitness-enhancing heterogeneity across an isogenic population of cells.

Three-Dimensional Chromatin Model of α-Globin Gene Locus in Differentially Activated States

The global architecture of cell nucleus and the spatial organization of chromatin play important roles in gene expression and nuclear function. By measuring the frequency of physical proximity between pairs of chromosomal regions on a locus scale, Chromosome Conformation Capture Carbon Copy (5C) technique revealed a wealth of information about the spatial organization of chromatin. However, the three-dimensional structure of the chromatin and how it changes at different activation states are largely unknown. We have developed an algorithm to reconstruct the three-dimensional structure of α-globin gene locus, which is expressed in K562 cell line and inactive in GM12878 cell line based on publicly available 5C interaction data [1]. Our reconstructed three-dimensional chromatin models show an open conformation in the active cells, whereas they fold into more compact conformations in inactive cells. Furthermore, our models not only can capture known long-range interactions, but can also predict new interactions that were not in the 5C data, but were experimentally observed in an independent study. Analysis of ensemble of model chromatin chains reveals an activation mechanism of α-globin gene that is highly influenced by the folding landscape and the spatial organization of the locus. Clusters of interacting regions suggest that the accessibility of specific regions of regulatory elements in three-dimensional space is essential for cells to maintain the necessary long-range interactions for gene activation.[1] Bau, D. et al. The three-dimensional folding of the α-globin gene domain reveals formation of chromatin globules. Nat Struct Mol Biol. 18, 107-114 (2011)

Stability of Inactive Components of Cathode Laminates for Lithium Ion Batteries at High Potential

The stability of inactive components in LIB (lithium ion batteries) electrodes upon exposure to high potentials can affect cell performance. A series of Li/ inactive component cells with aluminum, conductive carbon, and graphite as the inactive component were prepared and stored at high potential for one week. Electrochemical measurements and ex-situ surface analysis, including TEM (transmission electron microscopy), XPS (X-ray photoelectron spectroscopy), and FTIR (Fourier transform infrared spectroscopy), were conducted to investigate the stability of inactive components in the presence of LiPF6 in 3:7 ethylene carbonate (EC) and ethyl methyl carbonate (EMC) electrolyte at different potentials. The results show that all components are stable upon storage at 4.3 V. Storage at 4.6 or 4.9 V results in no aluminum corrosion, but limited decomposition on conductive carbon and greater decomposition on graphite. Storage at 5.3 V results in significant electrolyte oxidation to generate poly(ethylene carbonate) on the surface of all inactive electrodes and aluminum corrosion.

Virally-Mediated Versus Grazer-Induced Mortality Rates in a Warm-Temperate Inverse Estuary (Spencer Gulf, South Australia)

We investigated the seasonal dynamics of flow cytometrically-defined populations of viruses, heterotrophic bacteria, and the picoeukaryotic and prokaryotic phytoplankton at three sites in the temperate oligotrophic inverse estuary of Spencer Gulf (South Australia). We consistently identified two sub-populations of viruses, three sub-populations of heterotrophic bacteria, one population of picoeukaryotic phytoplankton and two populations of prokaryotic phytoplankton (cyanobacteria Prochlorococcus and Synechococcus). Both the cytometric community composition and the abundance of viruses, heterotrophic bacteria and both prokaryotic (Synechococcus and Prochlorococcus) and eukaryotic picophytoplankton were consistent with previous observations conducted in South Australian continental shelf waters. Noticeably LDNA bacteria (i.e. inactive or dormant cells) were consistently significantly the most abundant group of heterotrophic bacteria (totaling from 29% to 68% of total bacterial abundance) and were up to 10-fold more abundant than that previously reported in South Australian continental shelf waters, including the nearby Saint Vincent Gulf. These results suggest an overall low activity of the microbial community, and are consistent with previous evidence that LDNA cells may play a greater role in heterotrophic processes than HDNA cells in oligotrophic waters. In an attempt to further assess the qualitative and quantitative nature of the mortality of these organisms, we used a specific dilution assay to assess the relative contribution of viruses and microzooplankton grazers to the mortality rates of heterotrophic bacteria, and picoeukaryotic and prokaryotic phytoplankton. We consistently reported site-specific, population specific and sea-son-specific viral lysis and grazing rates of heterotrophic bacteria and the picoeukaryotic and prokaryotic (cyanobacteria Prochlorococcus and Synechococcus) phytoplankton across sites and seasons. Specifically, both viral lysis and micro-zooplankton grazing rates of heterotrophic bacteria were consistently relatively low across sites and seasons, even though their seasonality suggested an overall dominance of grazing over viral lysis in both summer and winter. In contrast, no seasonality is found in either lysis or grazing rates of prokaryotic and eukaryotic picophytoplankton, which are comparable to previous observations conducted in oligotrophic waters, suggesting the mortality dynamics of these populations is similar to those encountered in other oligotrophic waters. The observed patterns of mortality rates of heterotrophic bacteria and both prokaryotic and eukaryotic picophytoplankton are consistent with the low chlorophyll concentration and production previously observed in the waters of the Spencer Gulf.

Evaluation of Native Mesenchymal Stem Cells from Bone Marrow and Local Tissue in an Atrophic Nonunion Model

It has been reported that cells or growth factors might improve bone regeneration in nonunion. However, the intrinsic potential of the nonunion tissue to regenerate bone is still unclear; in particular, it is not known whether there are progenitor cells within the nonunion. Thus, in this study, a clinically relevant model has been developed to investigate the nature of the cells in atrophic nonunion tissue and to assess their regenerative potential. Seventeen Wistar rats underwent the procedure to induce an atrophic nonunion at the tibial midshaft by stripping the periosteum and endosteum as well as creating a small (1.0 mm) noncritical gap. The fracture was stabilized with an external fixator. The proliferation ability of bone marrow-derived cells and nonunion tissue-derived cells was determined using colony forming assays. The differentiation potential of nonunion tissue-derived cells was also investigated. Noncritical size defect nonunions were successfully induced in all of the animals. The typical characteristics of atrophic nonunions were demonstrated by radiography, micro-CT, and histology. An atrophic nonunion stimulated a systemic response in the bone marrow with an increase in bone marrow-derived mesenchymal stem cells. In contrast, cells derived from the nonunion gap were not able to form colonies. This indicated that the quiescent or inactive cells in the tissue at the atrophic nonunion gap could be induced to differentiate under osteogenic and chondrogenic conditions, but not under adipogenic conditions. Thus, these cells had the characteristics of osteochondral progenitor cells. Although there was an increase in the systemic response in the atrophic nonunion model, the intrinsic potential of local tissue from the atrophic nonunion site was impaired. The cells derived from nonunion tissue could be stimulated to differentiate into bone under appropriate conditions. This suggested that there are progenitor cells in atrophic nonunion tissue, which have an intrinsic ability to regenerate bone, but the microenvironment in the atrophic nonunion site might be retarding their function. Consequently, physical or biological agents (such as growth factors or cells) are needed to reactivate the endogenous progenitor cells to make them regenerate bone in atrophic nonunions.

Microglia – the brain’s busy bees

Recent years have seen significant changes in the way scientists view microglia and their role in health and disease. For decades, it was presumed that microglia were stationary, inactive immune cells in the brain, waiting for an immunologic insult to call them into action. In contrast, modern imaging techniques have revealed that microglia are constantly in motion, surveying their environment. Lineage tracing studies have led to the understanding that microglia are part of a larger family of cells, called tissue-resident macrophages, which arise from early yolk sac progenitors during embryogenesis and engraft nearly every organ in the body. Microglia, and all tissue-resident macrophages, rely on signaling through CD115 (the colony stimulating factor 1 receptor) for survival, primarily through the ligand, macrophage colony-stimulating factor. However, it is now understood that some microglia have a specific need for another CD115 ligand, Interleukin-34, which is only shared with Langerhans cells in the skin. In contrast to classical views, recent evidence suggests that the primary functions of microglia may occur during postnatal neurodevelopment and adult homeostasis, as absence or impairment of microglia results in a pathology separate from inflammatory immune function. In summary, these advances suggest that microglia might eventually be utilized or targeted to improve disease outcomes via encouraging or enhancing their health-promoting homeostatic functions.

Use of Fluorescein Diacetate (FDA) as a Single-Cell Probe of Metabolic Activity in Dinoflagellate Cultures

AbstractOur goal was to determine what percentage of cells remain metabolically active under environmentally relevant culture conditions. The approach combines epifluorescence microscopy and/or a standard bench-top fluorometer, plus the probe fluorescein diacetate (FDA). This stain is colorless in solution when added at a final concentration of 10 μM. It readily penetrates the cell membrane, and once inside the cell esterases cleave acetates to form fluorescein. Within 10 minutes, metabolically active cells become brilliantly yellow-green (fluorescence measured at 515–530 nm) while inactive cells do not fluoresce or are only weakly fluorescent. The FDA assay does not degrade the chlorophyll a fluorescence signal (>675 nm) nor does it interfere with normal motility or carbon uptake in most instances. Thirty clones of dinoflagellates were examined in this study. We found this assay useful to (1) optimize culture conditions and transfer schedules; (2) evaluate trauma of transfer into new media; (3) test for ...

Abstract C36: Is DHT involved in the action of TGF-β1 in epithelial ovarian cancer?

Abstract Ovarian cancer is the ninth most common female cancer worldwide and the fifth leading cause of cancer-related death among women in developed countries. In Chile, ovarian cancer is the second cause of death from gynaecological cancer. This disease is characterized by a silent course, late detection, high angiogenesis, with a poor response to therapy and a low survival. It is postulated that epithelial ovarian cancer (EOC) arises from the ovarian surface epithelium (OSE), but also it has been suggested that epithelial cells from fimbrae of the fallopian tubes could be implicated as the possible site of this malignancy, such as high-grade serous carcinoma. There are several hypotheses that intend to explain the origin of ovarian cancer and one of them postulate that sex steroid hormones have been involved in the aetiology and/or progression of some EOCs. In fact, androgens receptor (AR) is expressed in OSE cells and also in the majority of ovarian cancers. The microenvironment of ovarian cancer cells is enriched in androgens, and high levels of this steroid have also been reported in ascitic fluid; this condition supports the hypothesis that androgens, which are present in the ovarian stromal compartment, can produce neoplastic conversion of OSE and epithelial cells into de inclusion cyst and/or also be involved in ovarian cancer progression. Therefore, it is important to evaluate if dihydrotestosterone (DHT) is involved in the regulation of TGF-β1 receptors and also TGF-β1 signaling pathway, which is a potent inhibitor mechanism of cell proliferation, in EOC. This work was done with two types of experiments: 1) ex-vivo, with tissues of inactive normal ovaries and EOC poorly differentiated obtained from 10 patients from Hospital Clinico Universidad de Chile, with informed consent approved by Ethical Committee of the Institution, in order to evaluate the detection of AR by immunohistotochemistry (IHC). 2) In vitro experiments with human ovarian surface epithelium cell line (HOSE) and human epithelial ovarian cancer cell line (A2780). Both human cell lines are a good model that represents the epithelial cells from inactive normal ovaries and from EOC, respectively. These cells were stimulated with DHT and also with TGF-β1 during 72 h. The AR expression in cell lines was analysed by RT- PCR and immunocytochemistry (ICC). On the other hand, in both cell lines, TGF-β1R1 and TGF-β1R2 were detected by ICC and also in basal condition and with 10 and 100nM of DHT, by western-blot. In addition, we used an AR siRNA in order to know if DHT is involved in the TGF-β1R1 and/or in TGF-β1R2 expression in A2780 cell line. Our results show that AR protein levels in EOC and A2780 are significantly higher than inactive normal ovaries and HOSE cells, respectively (p<0.05). In basal condition, TGF-β1R1 protein levels in HOSE cells were higher than in EOC cells, but no changes were found in TGF-β1R2 protein levels in A2780. When cellular lines were treated with 100nM DHT, TGF-β1R1 and TGF-β1R2 protein levels decreased (p<0.05) with respect to the control condition only in A2780 cells. Besides, a 45% of AR silencing was obtained with AR siRNA (100 pmol) at 72 h in A2780 cells. Then, these cells were stimulated with 100 nM of DHT and it was obtained a 30% protein increase of the TGF-β receptors. These preliminary results suggest that DHT could be involved in TGF-β1 signaling pathway in epithelial ovarian cancer. This work has been support by grant FONDECYT # 1110372 (to CR) Citation Format: Karla Kohan, Cristian Poblete, Margarita Vega, Carmen Romero. Is DHT involved in the action of TGF-β1 in epithelial ovarian cancer? [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C36.

Dielectric monitoring and respirometric activity of a high cell density activated sludge

A novel method was developed to assess the viability of activated sludge present in a biological wastewater treatment process and signify its distinction from respirometric activity. The respirometric activity and viability of activated sludge at high cell density, such as typically encountered in membrane bioreactors, were investigated in batch and fed-batch systems. The method for measuring the viability of activated sludge was based on the sludge permittivity monitored online by a capacitive sensor. Results from permittivity measurement were compared with usual biological activity measurement through oxygen uptake rate determination. The similar downward trend was observed for both measurements. The respirometric activity and permittivity, respectively, reduced to 50% and 68% of initial value in the fed-batch system and 18% and 27% of initial value for the batch system which was due to quantitative and qualitative changes in the microbial culture in the activated sludge. The novel method allows to made distinction between viable versus dead and inactive versus active microbial cells in the activated sludge system and can be used for better and more efficient control of the biological processes.

Investigation into the physiological state of Escherichia coli K 12 cells under the action of TiO2 nanoparticles in acidic conditions

The physiological state of E. coli K12 bacterial cells under the action of TiO2 nanoparticles (0.04 g/L) was investigated with the method of staining by an acridine orange fluorescent dye in order to elucidate the mechanism of the antibacterial activity of nanoparticles. Experiments were performed in deionized water at initial pH 4.05–4.15, at which the surfaces of cells and nanoparticles carry opposite charges, without and with UV irradiation. The staining of the cells after incubation with TiO2 nanoparticles showed that both in the absence and under the action of UV irradiation, inactive cells were detected only in the composition of aggregates formed in the process of incubation and predominantly in their inner part. Active cells were found mainly in the composition of small aggregates and on the surface of the large aggregates, as well as single cells. The results obtained in this study allow suggesting that the compression due to the mutual attraction of the cells coated with TiO2 nanoparticles plays a significant role in the process of E. coli K12 cell inactivation.

Hyperspectral enhanced dark field microscopy for imaging blood cells

In this work, a novel methodology based on hyperspectral imagery with enhanced Darkfield microscopy for probing and characterizing changes in blood cell components was tested. Two main categories of blood cells were analyzed, red and white blood cells. Unique spectral signatures of ordinary and most common deformed morphologies of red blood cells were identified. Moreover, examination of white blood cells allowed to characterize and differentiate active from inactive cells. The findings indicate the ability of this technique to detect changes in light scattering property of blood cells due to their morphological properties Since pathological states can alterate the discocyte shape, this preliminary, but promising application of the hyperspectral analysis to blood cells can be useful to evaluate significant correlations of blood cell spectral features in healthy and pathological conditions. The combination of the quali- and quantitative spectral signatures of hyperspectral imaging microscopy with the information of the subject health conditions may provide a new tool for clinical applications.

Reliable detection of viable Salmonella enterica from inoculated chicken samples by fluorescent in situ hybridization (FISH) with vivification step

Inspection of food for the presence of Salmonella is a major concern in food industry. Many rapid, reliable and sensitive novel methods have been developed to detect Salmonella from food samples. However a method to detect live bacterial cells separately from dead cells yet to be developed. Although fluorescent in situ hybridization (FISH) has been identified as promising method in this regards, the viable count could be overestimated by FISH technique due to dead cells with some amount of non degraded rRNA. Some times it could be underestimated because of inactive but viable cells which not produce detectable amount of rRNA. The possibility of using antibiotic treatment step in FISH technique to overcome this problem was studied. Chicken samples inoculated by differently prepared mixtures of live and heat killed Salmonella enterica cultures were used to isolate bacterial cells with and without antibiotic treatment (Nalidixic acid 10μg/ml and Ciprofloxacin 1μg/ml for 2h at 370C). FISH was performed with Salmonella specific 23S rRNA probe Sal3, 5’-AATCACTTCACCTACGTG-3’ labeled with fluorescein isothiocyanate (FITC) at 5’ end. Cells observed with high intensity under fluorescent microscope were identified as live cells. Results of statistical analysis for antibiotic treated and untreated samples indicated that the introduction of antibiotic treatment step in FISH technique permitted a successful application to over come the problem associated with viable Salmonella enterica detection and quantification.DOI: http://dx.doi.org/10.4038/suslj.v11i1.5868 Sabaragamuwa University Journal 2012; V. 11 No. 1 pp 13-19

Cryopreservation Causes Genetic and Epigenetic Changes in Zebrafish Genital Ridges

Cryopreservation is an important tool routinely employed in Assisted Reproduction Technologies (ARTs) and germplasm banking. For several years, the assessment of global DNA fragmentation seemed to be enough to ensure the integrity of genetic material. However, cryopreservation can produce molecular alterations in key genes and transcripts undetectable by traditional assays, such modifications could interfere with normal embryo development. We used zebrafish as a model to study the effect of cryopreservation on key transcripts and genes. We employed an optimized cryopreservation protocol for genital ridges (GRs) containing primordial germ cells (PGCs) considered one of the best cell sources for gene banking. Our results indicated that cryopreservation produced a decrease in most of the zebrafish studied transcripts (cxcr4b, pou5f1, vasa and sox2) and upregulation of heat shock proteins (hsp70, hsp90). The observed downregulation could not always be explained by promoter hypermethylation (only the vasa promoter underwent clear hypermethylation). To corroborate this, we used human spermatozoa (transcriptionally inactive cells) obtaining a reduction in some transcripts (eIF2S1, and LHCGR). Our results also demonstrated that this effect was caused by freezing/thawing rather than exposure to cryoprotectants (CPAs). Finally, we employed real-time PCR (qPCR) technology to quantify the number of lesions produced by cryopreservation in the studied zebrafish genes, observing very different vulnerability to damage among them. All these data suggest that molecular alterations caused by cryopreservation should be studied in detail in order to ensure the total safety of the technique.

Identification of target genes of PAX3-FOXO1 in alveolar rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is a soft tissue sarcoma categorized into two major subtypes: alveolar RMS (ARMS) and embryonal RMS (ERMS). Most ARMS express the PAX3-FOXO1 (P3F) fusion oncoprotein generated by the 2;13 chromosomal translocation. In the present study, the downstream target genes of P3F were identified by analyzing two independent sets of gene expression profiles: primary RMS tumors and RD ERMS cells transduced with inducible P3F constructs. We found 34 potential target genes (27 upregulated and 7 downregulated) that were significantly and differentially expressed between P3F-positive and P3F-negative categories, both in primary RMS tumors and in the inducible P3F cell culture system. Gene ontology analysis of microarray data of the inducible P3F cell culture system employed indicated apoptosis, cell death, development, and signal transduction as overrepresented significant functional categories found in both upregulated and downregulated genes. Therefore, among the 34 potential target genes, the expression of cell death-related [Gremlin1, cysteine knot superfamily 1, BMP antagonist 1 (GREM1) and death-associated protein kinase 1 (DAPK1)] and development-related [myogenic differentiation 1 (MYOD1) and hairy/enhancer-of-split related with YRPW motif 1 (HEY1)] genes were further investigated. The differential expression of GREM1, DAPK1, MYOD1 and HEY1 was confirmed in independent tumors and inducible cell culture systems. The expression of GREM1, DAPK1 and MYOD1 were significantly upregulated; HEY1 was significantly downregulated in independent P3F-positive ARMS tumors and transcriptionally active P3F cells, compared to those in ERMS tumors and transcriptionally inactive P3F cells. This study identified target genes of P3F and suggested that four downstream targets (GREM1, DAPK1, MYOD1 and HEY1) can contribute to the biological activities of P3F involved in growth suppression or cell death and myogenic differentiation.

The Osteocyte: An Endocrine Cell … and More

Few investigators think of bone as an endocrine gland, even after the discovery that osteocytes produce circulating fibroblast growth factor 23 that targets the kidney and potentially other organs. In fact, until the last few years, osteocytes were perceived by many as passive, metabolically inactive cells. However, exciting recent discoveries have shown that osteocytes encased within mineralized bone matrix are actually multifunctional cells with many key regulatory roles in bone and mineral homeostasis. In addition to serving as endocrine cells and regulators of phosphate homeostasis, these cells control bone remodeling through regulation of both osteoclasts and osteoblasts, are mechanosensory cells that coordinate adaptive responses of the skeleton to mechanical loading, and also serve as a manager of the bone's reservoir of calcium. Osteocytes must survive for decades within the bone matrix, making them one of the longest lived cells in the body. Viability and survival are therefore extremely important to ensure optimal function of the osteocyte network. As we continue to search for new therapeutics, in addition to the osteoclast and the osteoblast, the osteocyte should be considered in new strategies to prevent and treat bone disease.