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Age-related changes of apoptotic cell death in human lymphocytes

Age-related changes of apoptotic cell death in human lymphocytes

ptotic cells in lymphocytes from aged controls ( ⌬ increase over baseline: old, 6.56 ⫾ 0.86% versus young, 4.33 ⫾ 0.42%; *, P ⫽ 0.014; Fig. 2c). Moreover, when all inves- tigated subjects were pooled for regression analysis, in- creasing age was correlated with percentage of apoptotic cells by regression analysis. Spontaneous apoptosis after 24 h increased 2.1-fold (slope ⫽ 0.061 ⫾ 0.02) between 15 and 93 years of age [Fig. 3, dotted line (- - -)]. There was a significant (***, P ⬍ 0.001, n ⫽ 85) correlation between age and the portion of apoptotic cells. To ascertain that these alterations in apoptotic cell death were not linked to alter- ations in the distribution of PBMC subpopulations, the same experiments were performed with activated lymphocytes consisting to over 95% of CD3 ⫹ cells. Lymphocytes, which are undergoing proliferation, can be triggered to pro- grammed cell death by withdrawal of IL-2. IL-2 is the most critical determinant in this process [23]. Activated lympho- cytes have been shown to be more sensitive to apoptosis than nontreated native cells. Basal apoptotic levels of acti- vated T cells correlated significantly with the donor age (*, P ⬍ 0.05, n ⫽ 52; Fig. 4; young, 9.99 ⫾ 1.05%, median 9.87% versus old, 13.29 ⫾ 1.04%, median 12.17%). In addition, spontaneous in vitro apoptosis after 24 h (under these conditions also called AICD: activation-induced cell death, that is triggered by IL-2 treatment followed by with- drawal) did as well significantly correlate with age (*, P ⬍ 0.05, n ⫽ 45; Fig. 4; young, 16.22 ⫾ 1.29%, median 15.23% versus old, 19.58 ⫾ 1.45%, median 19.15%). 3.2. Increased basal levels of ROS in aging
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OPUS 4 | Mechanisms of apoptotic cell death of lymphocytes in aging and in Alzheimer's disease

OPUS 4 | Mechanisms of apoptotic cell death of lymphocytes in aging and in Alzheimer's disease

First, it was essential to find out, if all cells of the PBMC (T-, B-, and NK-lymphocytes and monocytes) were effected or only some subsets show this impaired behaviour. Monocytes show in vitro an adherent form to plastic vessels, indicating that they are hardly detectable by flow cytometry, using polypropylene (PP) tubes. Staining with CD14 mAb, as a marker for myelotic cells, revealed that monocytes can be found in PBMC by flow cytometry only in traces, though monocytes/ macrophages are obvious visible in the microscope, before subjecting to the PP tubes (data not shown). Therefore, the flow cytometric-analyzed PBMC consist mainly of lymphocytes, namely T-, B-, and NK-lymphocytes. For these reasons, samples of AD patients, young and elderly controls were analyzed for apoptotic cell death for each lymphocytes subset. All subsets showed typical age-related changes, however, those changes were most prominent in T cells. In AD, elevated levels of basal apoptosis were evident in T and NK lymphocytes, B cell apoptosis was not altered in demented persons. Moreover, the sum of apoptotic cells in all subsets covers the amount detected with PI taking into account the percentage content in the PBMC (apoptosis in T cells of elderly controls: 1.08%; apoptosis in B cells of elderly controls: 5.03%; apoptosis of NK cells in elderly controls: 1.47%; apoptosis of T cells in AD patients: 1.54%; apoptosis of B cells in AD patients: 4.89%; apoptosis of NK cells in AD patients: 2.78%. à controls: (1.08% x 0.6596) + (5.032% x 0.1088) + (1.47% x 0.2436) = 1.618% apoptotic cells in PBMC by 7-AAD staining in elderly controls. AD: (1.54% x 0.6979) + (4.89% x 0.1034) + (2.78% x 0.2332) = 2.229% apoptotic cells in PBMC by 7-AAD staining in AD). The following table shows the determined apoptosis of PBMC with PI and the calculated data for PBMC determined with 7- AAD. It is evident that there variations between both methods. It should be noted that only a few identical individuals (patients and controls) were included in both studies. Most subjects were different, a propable reason for the deviation. However, AD-patients show elevated levels by both flow cytometric methods and by nucleosome ELISA (Eckert et al., 1998; 2001a).
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Uropathogenic Escherichia coli cause resistance to apoptotic cell death of infected cells by epigenetically suppressing BIM expression

Uropathogenic Escherichia coli cause resistance to apoptotic cell death of infected cells by epigenetically suppressing BIM expression

threonine and serine sites leads to cytoplasm sequestration and further degradation (Tzivion et al., 2011). Growth factor withdrawal or other factors inhibit AKT signaling, which consequently results in FOXOs nuclear accumulation and activation. In the present study, it has been shown that the UPEC virulence factor HlyA inhibits the AKT signaling pathway, consequently activating downstream FOXO proteins. Moreover, in the UPEC-elicited rat orchitis model, activation of FOXO1 and FOXO3 was documented by their dephosphorylation. The immunofluorescence data further confirmed that the expression of FOXO1 and FOXO3 was observed in the nuclei of testicular cells in the infected testis (Figure 7B, C). Additionally, in this experimental orchitis model, FOXOs are shown to be located in Sertoli cells, which are one of the most important somatic cell types in seminiferous tubules, playing an essential role in both supporting spermatogenesis and maintaining immune tolerance (Kaur et al., 2014). In the mock-infected testis, both FOXO1 and FOXO3 are barely detectable, likely due to the constitutive activation of the AKT signaling pathway. This observation is similar to the study by Goertz and colleagues, in which they demonstrated that FOXO1 was only detectable in undifferentiated spermatogonia, but not in the Sertoli cells or any other somatic cells of the mouse testis, suggesting FOXO1 plays an essential role in maintenance of spermatogonial stem cells and initiation of spermatogenesis (Goertz et al., 2011). The same group further showed that FOXO1 was expressed in undifferentiated spermatogonia in other mammals including humans (Tarnawa et al., 2013). Similar to the UPEC-infected testis, FOXOs are also activated after UPEC infection in isolated Sertoli cells, which has been further confirmed by an increase in DNA binding activity (shown in Figure 13). In addition, HDM did not activate FOXOs due to the lack of the virulence factor HlyA. These results are in agreement with a previous study, which showed that following AKT inhibition by the UPEC virulence factor HlyA, the phosphorylation of FOXO1 was decreased (Wiles et al., 2008).
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Apoptotic effects of TGFbeta superfamily members in isolated adult rat cardiomyocytes

Apoptotic effects of TGFbeta superfamily members in isolated adult rat cardiomyocytes

Apoptotic cells can be recognized by stereotypical morphological changes: the cell shrinks, shows deformation and loses contact to its neighbouring cells. Its chromatin condenses and marginates at the nuclear membrane, the plasma membrane is blebbing or budding, and finally the cell is fragmented into compact membrane-enclosed structures, called 'apoptotic bodies' which contain cytosol, the condensed chromatin, and organelles. The apoptotic bodies are engulfed by macrophages and thus are removed from the tissue without causing an inflammatory response. Those morphological changes are a consequence of characteristic molecular and biochemical events occurring in an apoptotic cell, most notably the activation of proteolytic enzymes, which mediate the cleavage of DNA into oligonucleosomal fragments as well as the cleavage of a multitude of specific protein substrates which usually determine the integrity and shape of the cytoplasm or organelles (Saraste et al, 2000). Apoptosis can be distinguesh from necrotic mode of cell- death in which the cells suffer a major insult, resulting in a loss of membrane integrity, swelling and disrupture of the cells. During necrosis, the cellular contents are released uncontrolled into the cells environment which results in damage of surrounding cells and a strong inflammatory response in the corresponding tissue (Leist et al, 2001).
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Characterisation of Anti-Apoptotic Signalling Pathways in Hepatocytes activated by alpha-Lipoic Acid and Atrial Natriuretic Peptide

Characterisation of Anti-Apoptotic Signalling Pathways in Hepatocytes activated by alpha-Lipoic Acid and Atrial Natriuretic Peptide

α-lipoic acid has also been described to promote apoptotic cell death: LA but no other antioxidant promotes apoptosis in retinal cell layers (269). Likewise, van de Mark et al. demonstrated LA-mediated apoptosis in transformed but not in normal cells, independent of the Fas signalling pathway (262). Furthermore, exposure to LA has been suggested to induce apoptosis by Fas in leukemic Jurkat cells but not in peripheral blood lymphocytes, with LA potentiating all effects following Fas activation, such as a rapid loss of cell thiols, an increase of intracellular Ca 2+ concentrations, and an activation of PKC and Caspase-3 (270). Besides, DHLA is also described to induce apoptosis in Jurkat T-cells (270). In addition, Casciari et al. determined that LA was not only toxic to different tumor cell lines, but also enhanced the cytotoxicity of ascorbate in tumors in a synergistic fashion (271). A possible reason for this synergy might be the ability of LA to modify the cellular redox status or to recycle ascorbate. Moreover, LA induced apoptosis in both, proliferating and non-proliferating cells, whereas non-proliferating cells are relatively resistant to ascorbate toxicity.
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The role of RIP-1 and cIAPs in apoptotic and non-apoptotic signalling via TLR3 and death receptors

The role of RIP-1 and cIAPs in apoptotic and non-apoptotic signalling via TLR3 and death receptors

The probable use of IAP antagonists, combined with TLR3 or death receptor agonists as treatment for tumours, should always relate to studied in primary keratinocytes in this thesis. Beside the obvious differences between primary keratinocytes and the model SCC cell lines, such as wild type p53 and the likely absence of chromosomal aberrations in primary keratinocytes (Boukamp, 2005), there are also several other differences. For example, primary keratinocytes have high levels of cFLIP expression and therefore are resistant to TRAIL-induced cell death (Leverkus et al., 2000). In contrast, the sensitivity of primary keratinocytes to TLR3 in this study was on the same level as in HaCaT (immortalised keratinocytes). An additional important difference of primary keratinocytes is that they express high levels of XIAP, which is also expressed by MET1 cells, but is absent in HaCaT and the derived cell line A5RT3. Similar to the data in the SCC models, primary keratinocytes were further sensitized to TLR3-induced cell death by the absence of IAPs, and cell death showed apoptotic features. Whereas blocking of caspases also revealed the cryptic necrotic pathway (Figure 32, blue field). This necrotic cell death was also dependent on RIP-1 kinase activity, duplicating the findings in the immortalised keratinocytes. Intriguingly and in contrast to the SCC cell lines, blocking of RIP-1 kinase functions in the presence and absence of IAPs led to an increase of apoptotic cell death, indicating the crucial relevance of RIP-1 kinase function for antiapoptotic protection of primary keratinocytes (Figure 32, blue field). Intriguingly the combined inhibition of RIP-1 and caspase activity could not grant full protection against TLR3-iduced cell death in the presence as well as in the absence of IAPs. This indicates that there are other TLR3 pro-death signalling pathways, which are not mediated by caspases or RIP-1 kinase function. One of the possibilities could be the activation of IFN regulatory factors (IRF) 3, 5 and 7 upon stimulation of TLR3 (Boo and Yang, 2010). Activation of these factors can be induced by binding of TRAF3 to TRIF and further association of TRAF3 into a complex with IKKε and TBK1 (Hacker and Karin, 2006). IRF5 is a strong transcription activator for IFN-α production, and IRF7 can induce IFN-α and also IFN-β preferentially (Boo and Yang, 2010). The IFNs in turn can induce apoptosis in various cell types (Chawla-Sarkar et al., 2003).
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Functional screening for anti-apoptotic genes using RNAi technology

Functional screening for anti-apoptotic genes using RNAi technology

The completion of the human genome sequence followed by initial functional analyses of the human transcriptome has resulted in the identification of novel genes, most of them uncharacterized or with poorly understood functions (Birney et al., 2007). Currently, for functional analysis of candidate genes, recombinant DNA or RNA molecules are widely used to over-express or to knock-down specific target genes in model cell cultures. When followed by an appropriate read out of the phenotype, such gene modulation approaches can be applied to identify gene functions related to certain cellular pathways (Sturzl et al., 2008). The aim of this study was to exploit the potential of loss-of-function screening to identify novel genes which are involved in apoptosis and cell survival. To reach this goal, we applied a functional genomics approach using RNAi technology in two different cell line models. A normal cell line, human embryonic kidney cells (HEK293T), and the tumorigenic MCF7 cell line were used for RNAi transfection and functional read out assays. Screening in HEK293T cells was carried out using short hairpin-coding constructs (shRNA) targeting 288 poorly characterized human genes. By measuring apoptosis as a readout of loss-of-function screening, CHMP5 was identified as a candidate for detailed functional and pathway analysis. Further investigation revealed an involvement of CHMP5 in apoptotic cell death and provided evidence on involvement of TNFR1 surface expression as an upstream and NF-  B signaling as a downstream affected factors. RNAi screening in breast cancer MCF7 cells using siRNAs targeting 107 selected genes over expressed in pNC identified PPP1R15B as a validated gene involved in survival and proliferation of MCF7 cells.
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Functional analysis of the Caspase-mediated cleavage of the pro-apoptotic tumor suppressor protein PAR-4

Functional analysis of the Caspase-mediated cleavage of the pro-apoptotic tumor suppressor protein PAR-4

PAR-4 can also bind to the pro-apoptotic kinase DLK, also known as zipper interacting protein kinase (ZIPK). DLK has been shown to interact and recruit death-associated protein 6 (Daxx) to promyelocytic leukemia (PML) nuclear bodies following induction of apoptosis with arsenic trioxide or interferon gamma. Interestingly, association of DLK and Daxx was accelerated by PAR-4, resulting in enhanced Caspase activity and apoptosis and suppression of each of these proteins reveals a decrease in apoptosis mediated by arsenic trioxide or interferon gamma [Kawai, Akira, and Reed 2003]. In parallel, PAR-4 was found in PML nuclear bodies, where it co-localizes with the pro- apoptotic protein THAP domain-containing protein 1 (THAP1) in primary endothelial cells, fibroblasts and in blood vessels in vivo [Roussigne et al. 2003]. Although the precise mechanism is unknown, forced PAR-4 gene expression also leads to the relocation of DLK from the nucleus to the cytoplasm and association to actin filaments, provoking reorganization of the cytoskeleton and an apoptotic phenotype [Page et al. 1999]. Moreover, binding of PAR-4 to actin filaments results in the generation of actin bundles in vitro and in vivo and is proposed to serve as a scaffold to recruit DLK, which then phosphorylates its substrate myosin light chain (MLC), upon induction of apoptosis [Vetterkind et al. 2005; Boosen et al. 2009; Vetterkind and Morgan 2009]. In a more recent study Alvarez and co-workers investigated the role of PAR-4 during apoptosis in diverse breast cancer models in mice and its relevance in tumor formation. PAR-4 was observed to be up-regulated upon oncogenic HER2/neu inhibition and treatment with chemotherapeutic drugs induced DLK-mediated phosphorylation of MLC in a PAR-4- dependent manner. Finally, they demonstrated that deregulated MLC phosphorylation is linked to multinucleation and ultimately apoptotic cell death due to cytokinesis failure [Alvarez et al. 2013].
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Cell mechanics and cell-cell interactions of fibroblasts from Dupuytren's Patient : Atomic Force Microscopy Investigation

Cell mechanics and cell-cell interactions of fibroblasts from Dupuytren's Patient : Atomic Force Microscopy Investigation

of epithelial cells, the biochemical expression of α-sma and type I and III collagen was induced in epithelial cell co-cultured fibroblasts (Morishima et al., 2001). These observations brought the knowledge of investigating the adhesion proteins involved in hetero-cellular interactions such as epithelial cell-fibroblasts interaction. For SCFS hetero-cellular studies, MDCK epithelial cells were attached on the cantilever and brought in contact with fibroblasts grown in monolayers in a Petri dish. This experimental set-up was chosen to measure the rupture forces of the N/N homophilic and E/N, E/OB and N/OB heterophilic bonds. Distinct peaks were not observed in the rupture force histograms (Fig. 5.3B, 5.3C and 5.3D), but the observed heterophilic bond results are able to be discussed with previous results. For example, an earlier SCFS study did not show any occurrence of heterophilic interactions between E- cadherin and N-cadherin (Panorchan et al., 2006b). Contrarily, a single molecule study shows the existence of such E/N cadherin heterophilic interactions (Prakasam et al., 2006) and in this study, we observed such E/N cadherin heterophilic interactions from the cell-cell adherens site. Presumably, shorter contact/dwell times (millisecond) used in the former studies could be the reason for not recognizing heterophilic interactions as found in our study (Prakasam et al., 2006). In standard experimental settings, shorter contact times between the cells in the petri dish and on the cantilever were used to prevent nonspecific binding. Deliberately, we chose a different experimental design with longer contact time of 2 sec. in the SCFS setup which enabled us to follow both homophilic and heterophilic cadherin interactions. Despite of the changed protocol, distinct peaks could not be resolved in the histograms. This might be due to the possibility that N/N and E/N rupture forces share similar values. In case of DF-MDCK, no distinct peaks of E/OB and N/OB were seen which could be due to similar rupture forces. This leads to the question if single molecule kinetic studies using AFM or optical tweezers are suitable to measure homophilic and heterophilic cadherin pairs.
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Single-cell arrays for dynamic analysis of cell processes

Single-cell arrays for dynamic analysis of cell processes

If a few hundrets of target cells are covered by 1 microliter of fluid, each carrying 360,000 copies of CD123 on its surface, then this would amount to a few million molecules of CD123 initially exposed on the surface of this pool of targets facing 600 million molecules of triplebody. Consequently, under these saturating concentrations, an approximately 100-fold (or greater) numerical excess of triplebody in the fluid phase is present over the number of target antigens on the target cell surface. Under these circumstances, most CD123 molecules on the surface will be occupied by a triplebody molecule and will be internalized within approximately 30 to 60 minutes, a process called “down-modulation of the target antigen”. Some of the internalised CD123 molecules will be recycled to the surface, and new synthesis of CD123 will also occur, but these resurfacing molecules will again be rapidly re-internalised, because the 100-fold excess of triplebody in the fluid phase will not be exhausted. This recycling and re-internalisation will occur continuously over the 16 hour measurement period, and it is therefore not obvious, how this process could contribute to the observed reduction of reaction rates after the maximum has been reached. As only less than half of the target cells are killed over the entire reaction period, it is difficult to conceive that the number of available target cells, which was reduced by less than 2-fold over this time, would explain the observed 2-fold drop in the rate of lysis. More likely, in addition to the contribution by the loss of target cell numbers, the targets must also be in a state of “preparedness” for lysis, and this preparedness may decrease with time in a manner influenced by the concentration of the triplebody. A mechanistic explanation for the decrease in reaction rates after reaching the observed maximum a few hours into the reaction interval is presently not obvious. If the explanation offered above for the increase in rates during the first few hours of the observation interval (on the way to the maximum) is correct, namely that a “kinetic boosting” occurs with a concurrent shortening of the time interval between successive lytic events of a single NK cell, than the observed slowing down of the reaction rates after reaching the maximum may be accompanied by a corresponding “kinetic dampening” of the NK cells, i.e. an increase in the median length of time between two consecutive lytic events of a single NK cell. The time-resolved SSC method presented here is sufficiently powerful to permit a direct experimental test of the validity of this hypothesis.
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Segmentation of cytological stained cell areas and generation of cell boundaries in complex shaded cell clusters

Segmentation of cytological stained cell areas and generation of cell boundaries in complex shaded cell clusters

It is very challenging to separate the individual cells within a cell cluster obvious from biological reasons. To achieve this, we first calculate the center of gravity for each cell core in the cluster. Then we determine the shortest path beginning from the most upper left center of gravity to the nearest next center and so on (fig. 8). For the calculation of the shortest path we use the A*-algorithm, as described in [6]. This algorithm is often used in game development. When calculating the shortest paths the algorithm has to take care of holes within the cell cluster. Possible obstacles, in our case holes must be avoided by the path. The basic idea of the algorithm is a cost function which is calculated for all neighboring pixels. The calculated cost from the intermediate point at position (x,y) to the starting point and the estimated cost from the same point to the endpoint are calculated by g(x,y) and h(x,y) respectively. The sum of the two cost functions is the total cost function f(x,y). That set of points {(x,y)} belong to the shortest path which minimizes f(x,y). We use the city block distance in order to reduce computing time.
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Cell polarisation in geometry

Cell polarisation in geometry

the interaction with the specific geometry. In case of different cytoplasmic states, which can originate at the membrane, a completely homogeneous steady state does not necessarily exist. For example, when considering an inactive state of proteins which is induced at the membrane and reactivation occurs in the cytosol. In this case, the membrane is like a source of inactive proteins which degrade back to their active state while diffusing through the cytosol. For an illustration see Fig. 3.1 (A): here, proteins become inactive for binding on the membrane. They diffuse through the cytosol where they reactivate according to some constant rate. Thus, the concentration gradiates from a very low concentration of active proteins close to the membrane to a high concentration in the cytosolic bulk far away from the membrane. Mathematically this is described as a source degradation problem [15]. Cell biological examples for such a scenario are proteins which undergo a nucleotide exchange from NDP- to NTP-bound, such as MinD in E. coli [35] and Cdc42 in yeast [85], and also phosphorylation on the membrane such as among PAR [86] counteracted with dephosphorylation in the cytosol. Cytoplasmic gradients not only exhibit themselves a (weak) pattern but are able to influence membrane patterns significantly [32, 35, 36], see also chapter 4. Pattern formation on cellular membranes is driven by positive or/and negative feedback loops of membrane attachment (including dimerisation) and detachment [38, 39]. The attachment and detachment processes, however, depend on the availability of proteins which can attach or dettach. This, in turn, depends also on cytosolic gradients. For illustration see Fig. 3.1 (B): Thalmeier et al. could show that in an elliptical geometry the concentration profiles of binding inactive (top) and active (middle) MinD are associated with a bipolar MinD pattern on the membrane (bottom). Furthermore, in this work (see Fig. 3.1 (C-F) and for details see also chapter 4), it is shown that in two-dimensional elliptical geometry the depth of an inactive layer of proteins strongly impacts pattern formation and alignment. This depth can either be altered by the speed of cytosolic diffusion, i.e. how far does an inactive protein travel given some time interval, or by the time until reactivation, i.e. for how long can it travel before it is active for binding again. In Fig. 3.1 (C,E) reactivation is fast which results in a vanishingly thin inactive layer. For pattern formation based on mutually antagonistic membrane detachment and inactivation, this results into the highest recycling rates of proteins at the cell poles and thus a short-axis polarised cell. For slow reactivation as in Fig. 3.1 (D,F) the system exhibits a much wider inactive layer. In the polar regions least active proteins are available, so recycling is weakest here and strongest at mid-cell. Thus, in equilibrium the pattern is long-axis polarised.
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CAR T-cell therapy in diffuse large B-cell lymphoma

CAR T-cell therapy in diffuse large B-cell lymphoma

CAR T-cell therapy is “en vogue” due to very promis- ing response data in relapsed and refractory DLBCL and 50–70% of patients will be alive after 12 month trials. This has been confirmed in “real-world” data including patients not eligible for trials due to several comorbidities [5, 6, 9]. Despite the high efficacy of CAR T-cell therapy with CR rate up to 50%, which is in contrast to poor outcome after conventional salvage immune-chemotherapy, results have to be interpreted with caution [17]. So far, only data of single-arm phase II trials with highly selected patients and short ob- servation times are available. Even “real-world” data do not really improve the information quality, as data were provided by only a few highly experienced cen- ters. No randomized data comparing ASCT or allo- HSCT are available so far. Therefore, no recommen- dation can be given in patients relapsing or refractory to first line therapy if transplant eligible. Clinical trials comparing ASCT with CAR T-cell therapy are currently being initiated.
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Characterization of anti-apoptotic protein
family members (Bax inhibitor-1 and Lifeguard)
in Hydra vulgaris

Characterization of anti-apoptotic protein family members (Bax inhibitor-1 and Lifeguard) in Hydra vulgaris

Also it is reported that BI-1 plays a key role in the regulation of intracellular ROS, which are highly reactive oxygen containing molecules and cause oxidative stress and induce apoptosis. ROS are produced at the ER by the NADPH-P450 reductase (NPR), phospholipids and the microsomal monooxygenase system composed of cytochrome P450 (CYP) members (Nieto et al., 2002; Robinson et al., 2011). BI-1 is able to regulate ER ROS production in different ways. Firstly, over expression of BI-1 in cells increased activation of the redox-sensitive transcription factor, i.e. Nrf-2 and controls/ increase the expression of different antioxidant enzymes like Heme-Oxygenase-1(see also Fig. 4A). Heme-Oxygenase-1 (HO-1) can block ROS activity and is expressed to counteract ROS accumulation and in this way promoting cell survival (Lee et al., 2007). Also BI-1 can interact through its C-terminus with NPR and destabilization the NPR-CYP2E1 complex and directly inhibit the formation of ROS by blocking electron transfer (Kim et al., 2009) (see also Fig. 4A).
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Gutenberg Open Science: Functional characterization of modified vaccinia virus Ankara-encoded anti-apoptotic proteins

Gutenberg Open Science: Functional characterization of modified vaccinia virus Ankara-encoded anti-apoptotic proteins

complete replication cycle, each gene class is responsible for initiating the expression of the upcoming class by encoding specific transcription factors. In return, the upcoming expression of one class is responsible for the transcriptional shutdown of the former class (Figure 2.3). The structural proteins finally assemble with discrete membrane structures (8), in which one DNA unit is packaged to form an immature virion (IV; 9). After a further maturation step, the first form of an infectious virion is built (10). This mature virion (MV) is wrapped in the trans-golgi network to gain additional membrane layers, then denoted as wrapped virion (WV; 11). WVs are then transported along the microtubule network towards the periphery of the cell (12), where fusion of the most outer viral envelope with the plasma membrane takes place. These extracellular virions (EV), now wrapped by two membrane layers, are either released from the cell or remain attached at the cell membrane (13). Attached EVs induce the polymerization of actin tails to expose the virion particles, which seems to be an efficient mechanism to infect neighbouring cells. Although the whole replication cycle takes place exclusively in the cytoplasm of infected cells, some nuclear and cytoplasmic cellular factors have already been described to be involved in transcription and morphogenesis.
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Cell-cell communication via LuxR solos in Photorhabdus species

Cell-cell communication via LuxR solos in Photorhabdus species

It is well recognized that bacteria communicate via small diffusible molecules, a process termed quorum sensing. The best understood quorum sensing systems are those that use acylated homoserine lactones (AHLs) for communication. The prototype of those systems consists of a LuxI-like AHL synthase and a cognate LuxR receptor that detects the signal. However, many proteobacteria possess LuxR receptors, yet lack any LuxI-type synthase, and thus these receptors are referred to as LuxR orphans or solos. In addition to the well-known AHLs, little is known about the signaling molecules that are sensed by LuxR solos. Here, we describe a novel cell-cell communication system in the insect and human pathogen Photorhabdus asymbiotica. We identified the LuxR homolog PauR to sense dialkylresorcinols (DARs) and cyclohexanediones (CHDs) instead of AHLs as signals. The DarABC synthesis pathway produces the molecules, and the entire system emerged as important for virulence. Moreover, we have analyzed more than 90 different Photorhabdus strains by HPLC/MS and showed that these DARs and CHDs are specific to the human pathogen P. asymbiotica. On the basis of genomic evidence, 116 other bacterial species are putative DAR producers, among them many human pathogens. Therefore, we discuss the possibility of DARs as novel and widespread bacterial signaling molecules and show that bacterial cell-cell communication goes far beyond AHL signaling in nature.
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Schwann cell precursor: a neural crest cell in disguise?

Schwann cell precursor: a neural crest cell in disguise?

Consistent with their name, SCPs generate all types of peripheral glial cells found in the adult body, including myelinating, non- myelinating (Remak) and terminal Schwann cells, as well as other not well-studied subtypes, including specialized glial cells in various sensory skin end organs. Di fferentiation of SCPs into Schwann cells, their largest derivative, includes a transitory state of an immature Schwann cell and partly resembles di fferentiation of oligodendrocytes in the central nervous system (reviewed by Kastriti and Adameyko, 2017 ). Such similarity stimulated discussions about the evolutionary connections between CNS and PNS glial cells types. For instance, it is possible that the gene expression module responsible for myelination evolved primarily at the periphery of the body to provide faster transmission of sensory and motor signals in animals, increasing their size. If so, the module could have been later co-opted into the CNS to increase the speed of complex computations. Adult Schwann cells do not exhibit any stem-like populations to the best of our knowledge and instead rely on processes resembling de-di fferentiation in case of nerve damage ( Jessen and Mirsky, 2016 ). In such cases, mouse adult Schwann cells located at the site of nerve damage can generate pigmented spots and have been con firmed to be the origin (both myelinating and non-myelinating subtypes) of local melanocytes ( Adameyko et al., 2009; Rizvi et al., 2002 ). Interestingly, in continu- ously growing mouse incisors, adult peripheral glial cells are periodi- cally recruited into the dental mesenchymal stem cell niche, giving rise to large amounts of odontoblasts and pulp cells during the mouse incisor tooth self-renewal process ( Kaukua et al., 2014 ). Similarly, extrinsic nerves with associated SCPs are capable of re-supplying the postnatal growing gut with progenitors of enteric neurons ( Uesaka et al., 2015 ), whereas trauma to the mature enteric ganglia induces the di fferentiation of resident glial populations into neuronal components ( Laranjeira et al., 2011 ).
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IDEAL-Cell, a High Temperature Innovative Dual mEmbrAne Fuel-Cell

IDEAL-Cell, a High Temperature Innovative Dual mEmbrAne Fuel-Cell

The novel IDEAL-Cell concept raises many challenges and fundamental questions, some of which emerged through our first experiments (such as the threshold effect seen in the impedance fingerprint of what seems to be the formation of water, and the exact nature of the recombination mechanism occurring at the central membrane). It should be emphasized that before SOFC prototypes emerged in the pre-market segment, as is the case today, many decades were spent on extensive research in electrochemistry, material science, solid state chemistry and chemistry. IDEAL-Cell is only 1 year old, and within this short lifetime a mere abstract concept has become a real fuel cell. However, an enormous amount of work has still to be done to bring IDEAL-Cell to the level of the mature SOFC systems.
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Metabolic, anti-apoptotic and immune evasion strategies of primary human myeloma cells indicate adaptations to hypoxia

Metabolic, anti-apoptotic and immune evasion strategies of primary human myeloma cells indicate adaptations to hypoxia

ess of newly synthesized proteins. Multiple myeloma is char- acterized by aberrantly glycosylated immunoglobulins (89), which are synthesized in high amounts by MM cells. Most importantly, glutamine can also be interconverted into aspar- agine, which might be used for the import of other amino acids via reciprocal exchange (90); levels of glutamine-hydro- lyzing asparagine synthetase ASNS and transporter proteins involved in these processes, SLC1A4 and SLC1A5, were found significantly increased in MM high cells (Fig. 4A). Results of the targeted analyses of amino acids further point to the likelihood that BM fibroblasts may supply MM cells with sev- eral of the necessary amino acids, probably in exchange for asparagine (Figs. 4B and 4C). This view is supported by the fact that BM stromal cells such as fibroblasts are widely used as feeder layers for MM cells in cell culture experiments (91). Remarkably, asparagine synthesis has been associated with survival of glutamine-dependent tumor cells, and expression of ASNS was correlated with poor prognosis in several tumors (92). Studies have indeed suggested good anti-myeloma ac- tivity of asparaginase (93).
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Anticancer Activity of Fascaplysin against Lung Cancer Cell and Small Cell Lung Cancer Circulating Tumor Cell Lines

Anticancer Activity of Fascaplysin against Lung Cancer Cell and Small Cell Lung Cancer Circulating Tumor Cell Lines

DNA damage response is triggered when sensor proteins ATM (ataxia telangiectasia mutated) and ATR (also called ataxia telangiectasia and Rad3-related protein) detect structural distortions or breaks [31]. After DNA damage, CHK2 is phosphorylated by ATM on the priming site T68, and in turn, phosphorylates more than 24 proteins to induce apoptosis, DNA repair, or tolerance of the damage [32]. In wildtype cells, CHK2 phosphorylates Rb which enhances the formation of the transcriptionally-inactive pRb/E2F-1 complex causing G1/S arrest and suppression of apoptosis. Pronounced activation of CHK-2 in NCI-H526 and A549 cells indicates direct damage of DNA by fascaplysin and activation of the corresponding cellular responses in both cell lines. The cyclic AMP response element-binding protein (CREB) initiates transcriptional responses associated with cell survival to a wide variety of stimuli following its phosphorylation on Ser-133. Whereas fascaplysin treatment resulted in decreased phosphorylation of CREB in NCI-H526 cells, this transcription factor is hyperphosphorylated in A549 cells, possibly indicating anti- and pro-survival signaling, respectively [33,34]. Furthermore, cisplatin-induced activation of FAK has been linked to increased chemoresistance in ovarian cancer cells and FAK inhibitors induce tumor cell apoptosis [35]. Activated FAK forms a complex with Src family kinases and seems to provide a prosurvival signal in NCI-H526 cells, in contrast to fascaplysin-treated A549 cells [36]. In addition, overexpression of Src in cancer accelerates metastasis and is responsible for chemoresistance via multiple downstream signaling pathways, concerning Akt, MAPKs, STAT3, cytokines, etc. [37]. Therefore, activation of a
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