Double in situ hybridization in combination with digital image analysis: A new approach to study interphase chromosome topography

Double in situ hybridization with mercurated and biotinylated chromosome specific DNA probes in combination with digital image analysis provides a new approach to compare the distribution of homologous and nonhomologous chromosome targets within individual interphase nuclei. Here we have used two DNA probes representing tandemly repeated sequences specific for the constitutive heterochromatin of the human chromosomes 1 and 15, respectively, and studied the relative arrangements of these chromosome targets in interphase nuclei of human lymphocytes, amniotic fluid cells, and fibroblasts, cultivated in vitro. We have developed a 2D-image analysis approach which allows the rapid evaluation of large numbers of interphase nuclei. Models to test for a random versus nonrandom distribution of chromosome segments are discussed taking into account the three-dimensional origin of the evaluated 2D-distribution. In all three human diploid cell types the measurements of target-target and target-center distances in the 2D-nuclear image revealed that the labeled segments of the two chromosomes 15 were distributed both significantly closer to each other and closer to the center of the nuclear image than the labeled chromosome 1 segments. This result can be explained by the association of nucleolus organizer regions on the short arm of chromosome 15 with nucleoli located more centrally in these nuclei and does not provide evidence for a homologous association per se. In contrast, evaluation of the interphase positioning of the two chromosome 1 segments fits the random expectation in amniotic fluid and fibroblast cells, while in experiments using lymphocytes a slight excess of larger distances between these homologous targets was occasionally observed. 2D-distances between the labeled chromosome 1 and 15 segments showed a large variability in their relative positioning. In conclusion our data do not support the idea of a strict and permanent association of these homologous and nonhomologous targets in the cell types studied so far

Challenges and status: Single-use bioreactors for microbial processes

Nowadays single-use bioreactors are fully accepted in the biopharmaceutical industry. Reactors up to 2000L working volume are commonly used. However, these bioreactors are limited in terms of mass-transfer and mixing capabilities and therefore only suited for application in mammalian cell culture. Single-use processing offers many benefits like cost reduction, flexibility, reduction of contamination risks, etc. These benefits apply for both microbial processes as for mammalian processes. Additionally, for the use of marine microorganisms the application of single-use bioreactors (SUB) offers a possibility to circumvent problem of corrosion which occurs in steel bioreactors due to the high chloride ion concentration in the media when the early stage of process development or the need for multi-purpose devices does not allow the investment in process-specific infrastructure. However, marine production processes can demand for high gas mass transfer rates, e.g. in the case of Crypthecodinium cohnii, a heterotrophic algae applied for the production of the polyunsatured fatty acid docosahexaenoic acid. C. cohnii cells are highly sensitive to shear-forces. In general, unfavorable cultivation conditions lead to a high batch-to-batch variation, and thus to a random process development and optimization. In this paper we describe experience of the use of different single use bioreactors for the high cell density cultivation of C. cohnii and other microorganisms. Specific parameters we looked at were the gas transfer efficiency as an important parameter for high cell and product yields as well as the opportunity for expansion of the culture over a wide volume range. Among various SUBs, which were tested, only the 2-D wave-mixed CELL-tainer® showed a high oxygen mass transfer at comparably low shear forces, and hence provided a very vital culture.1, 2 In order to broaden the range of the working volume, expansion channel blocks were applied, which allow performing cultivations from 150 ml to 20 L without reinocculation. However, the scalability of a wave-mixed system is challenging due to the restricted knowledge of classical engineering parameters. Therefore, the physiologic and morphologic constitution of the cells was considered to prove the suitability of the SUBs at scales from the mL to the 120 L range. Therefore a novel on-line photo-optical instrument (SOPAT) for the analysis of cell shapes, and lipid droplet accumulation was applied. It allowed a direct insight into the stage of growth, population homogeneity, and fatty acid production. Although it is hardly feasible to maintain identical cultivation conditions from the µL to the m³ scale, the combination of engineering parameters and process analytical tools led to the overall achievement of suitable cultivation conditions. The presented on-line method in relation to the developmental strategy over different scales is relevant for the development of plant and other cell culture processes, while contributing to reduced development times and costs

Miniaturized Wide-Range Field-Emission Vacuum Gauge

Miniaturized vacuum gauges (MVGs) for the measurement range 5.7x10-7 to 1.1x10-2 mbar were fabricated in a self-aligned approach using focused ion beam (FIB) nanomachining and reactive ion etching (RIE). The MVG consists of two properly insulated electrodes integrated on top of an atomic force microscopy (AFM) tip, forming a coaxial embodiment. The special design enables us to vary the cathode-anode separation and the turn-on voltage changes accordingly. The experiments show that the MVGs operate at low bias potential and demonstrate very good I-P dependence over a wide pressure range

Targeted inhibition of protein synthesis renders cancer cells vulnerable to apoptosis by unfolded protein response

AbstractCellular stress responses including the unfolded protein response (UPR) decide over the fate of an individual cell to ensure survival of the entire organism. During physiologic UPR counter-regulation, protective proteins are upregulated to prevent cell death. A similar strategy induces resistance to UPR in cancer. Therefore, we hypothesized that blocking protein synthesis following induction of UPR substantially enhances drug-induced apoptosis of malignant cells. In line, upregulation of the chaperone BiP was prevented by simultaneous arrest of protein synthesis in B cell malignancies. Cytotoxicity by immunotoxins—approved inhibitors of protein synthesis—was synergistically enhanced in combination with UPR-inducers in seven distinct hematologic and three solid tumor entities in vitro. Synergistic cell death depended on mitochondrial outer membrane permeabilization via BAK/BAX, which correlated with synergistic, IRE1α-dependent reduction of BID, accompanied by an additive fall of MCL-1. The strong synergy was reproduced in vivo against xenograft mouse models of mantle cell lymphoma, Burkitt’s lymphoma, and patient-derived acute lymphoblastic leukemia. In contrast, synergy was absent in blood cells of healthy donors suggesting a tumor-specific vulnerability. Together, these data support clinical evaluation of blocking stress response counter-regulation using inhibitors of protein synthesis as a novel therapeutic strategy.</jats:p

Recruitment of the Linear Ubiquitin Chain Assembly Complex Stabilizes the TNF-R1 Signaling Complex and Is Required for TNF-Mediated Gene Induction

TNF is a key inflammatory cytokine. Using a modified tandem affinity purification approach, we identified HOIL-1 and HOIP as functional components of the native TNF-R1 signaling complex (TNF-RSC). Together, they were shown to form a linear ubiquitin chain assembly complex (LUBAC) and to ubiquitylate NEMO. We show that LUBAC binds to ubiquitin chains of different linkage types and that its recruitment to the TNF-RSC is impaired in TRADD-, TRAF2-, and cIAP1/2- but not in RIP1- or NEMO-deficient MEFs. Furthermore, the E3 ligase activity of cIAPs, but not TRAF2, is required for HOIL-1 recruitment to the TNF-RSC. LUBAC enhances NEMO interaction with the TNF-RSC, stabilizes this protein complex, and is required for efficient TNF-induced activation of NF-κB and JNK, resulting in apoptosis inhibition. Finally, we demonstrate that sustained stability of the TNF-RSC requires LUBAC's enzymatic activity, thereby adding a third form of ubiquitin linkage to the triggering of TNF signaling by the TNF-RSC. © 2009 Elsevier Inc. All rights reserved