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Mutation of the LMNA gene, encoding nuclear lamin A and lamin C (hereafter lamin A/C), is a common cause of familial dilated cardiomyopathy (DCM). Among Finnish DCM patients, the founder mutation c.427T>C (p.S143P) is the most frequently reported genetic variant. Here, we show that p.S143P lamin A/C is more nucleoplasmic and soluble than wild-type lamin A/C and accumulates into large intranuclear aggregates in a fraction of cultured patient fibroblasts as well as in cells ectopically expressing either FLAG- or GFP-tagged p.S143P lamin A. In fluorescence loss in photobleaching (FLIP) experiments, non-aggregated EGFP-tagged p.S143P lamin A was significantly more dynamic. In in vitro association studies, p.S143P lamin A failed to form appropriate filament structures but instead assembled into disorganized aggregates similar to those observed in patient cell nuclei. A whole-genome expression analysis revealed an elevated unfolded protein response (UPR) in cells expressing p.S143P lamin A/C. Additional endoplasmic reticulum (ER) stress induced by tunicamycin reduced the viability of cells expressing mutant lamin further. In summary, p.S143P lamin A/C affects normal lamina structure and influences the cellular stress response, homeostasis and viability.

Quantum repeaters - fundamental building blocks for long-distance quantum communication - are based on the interaction between photons and quantum memories. The photons must fulfil stringent requirements on central frequency, spectral bandwidth and purity in order for this interaction to be efficient. We present a design scheme for monolithically integrated resonant photon-pair sources based on parametric down-conversion in nonlinear waveguides, which facilitate the generation of such photons. We investigate the impact of different design parameters on the performance of our source. The generated photon spectral bandwidths can be varied between several tens of MHz up to around $1\,$GHz, facilitating an efficient coupling to different memories. The central frequency of the generated photons can be coarsely tuned by adjusting the pump frequency, poling period and sample temperature and we identify stability requirements on the pump laser and sample temperature that can be readily fulfilled with off-the-shelve components. We find that our source is capable of generating high-purity photons over a wide range of photon bandwidths. Finally, the PDC emission can be frequency fine-tuned over several GHz by simultaneously adjusting the sample temperature and pump frequency. We conclude our study with demonstrating the adaptability of our source to different quantum memories., 10 pages, 8 figures

// Harald Herrmann 1 , Katharina Blatt 2 , Junwei Shi 3 , Karoline V. Gleixner 2 , Sabine Cerny-Reiterer 1 , Leonhard Mullauer 4 , Christopher R. Vakoc 3 , Wolfgang R. Sperr 1,2 , Hans-Peter Horny 6 , James E. Bradner 5 , Johannes Zuber 3,7 , Peter Valent 1,2 1 Ludwig Boltzmann Cluster Oncology, Vienna, Austria; 2 Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Austria; 3 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA; 4 Department of Pathology, Medical University of Vienna, Austria; 5 Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA; 6 Institute of Pathology, Ludwig-Maximilians-University, Munich, Germany; and 7 Research Institute of Molecular Pathology (IMP), Vienna, Austria. Correspondence: Peter Valent, email: // Keywords : AML, leukemic stem cells, BRD4, JQ1, targeted therapy Received : November 02, 2012, Accepted : November 26, 2012, Published : November 27, 2012 Abstract Acute myeloid leukemia (AML) is a life-threatening stem cell disease characterized by uncontrolled proliferation and accumulation of myeloblasts. Using an advanced RNAi screen-approach in an AML mouse model we have recently identified the epigenetic ‘reader’ BRD4 as a promising target in AML. In the current study, we asked whether inhibition of BRD4 by a small-molecule inhibitor, JQ1, leads to growth-inhibition and apoptosis in primary human AML stem- and progenitor cells. Primary cell samples were obtained from 37 patients with freshly diagnosed AML (n=23) or refractory AML (n=14). BRD4 was found to be expressed at the mRNA and protein level in unfractionated AML cells as well as in highly enriched CD34 + /CD38 - and CD34 + /CD38 + stem- and progenitor cells in all patients examined. In unfractionated leukemic cells, submicromolar concentrations of JQ1 induced major growth-inhibitory effects (IC 50 0.05-0.5 µM) in most samples, including cells derived from relapsed or refractory patients. In addition, JQ1 was found to induce apoptosis in CD34 + /CD38 − and CD34 + /CD38 + stem- and progenitor cells in all donors examined as evidenced by combined surface/Annexin-V staining. Moreover, we were able to show that JQ1 synergizes with ARA-C in inducing growth inhibition in AML cells. Together, the BRD4-targeting drug JQ1 exerts major anti-leukemic effects in a broad range of human AML subtypes, including relapsed and refractory patients and all relevant stem- and progenitor cell compartments, including CD34 + /CD38 - and CD34 + /CD38 + AML cells. These results characterize BRD4-inhibition as a promising new therapeutic approach in AML which should be further investigated in clinical trials.

The intermediate filament protein desmin is an essential component of the extra-sarcomeric cytoskeleton in muscle cells. This three-dimensional filamentous framework exerts central roles in the structural and functional alignment and anchorage of myofibrils, the positioning of cell organelles and signaling events. Mutations of the human desmin gene on chromosome 2q35 cause autosomal dominant, autosomal recessive, and sporadic myopathies and/or cardiomyopathies with marked phenotypic variability. The disease onset ranges from childhood to late adulthood. The clinical course is progressive and no specific treatment is currently available for this severely disabling disease. The muscle pathology is characterized by desmin-positive protein aggregates and degenerative changes of the myofibrillar apparatus. The molecular pathophysiology of desminopathies is a complex, multilevel issue. In addition to direct effects on the formation and maintenance of the extra-sarcomeric intermediate filament network, mutant desmin affects essential protein interactions, cell signaling cascades, mitochondrial functions, and protein quality control mechanisms. This review summarizes the currently available data on the epidemiology, clinical phenotypes, myopathology, and genetics of desminopathies. In addition, this work provides an overview on the expression, filament formation processes, biomechanical properties, post-translational modifications, interaction partners, subcellular localization, and functions of wild-type and mutant desmin as well as desmin-related cell and animal models.

Together with actin filaments and microtubules, intermediate filaments (IFs) are the basic cytoskeletal components of metazoan cells. Over 80 human diseases have been linked to mutations in various IF proteins to date. However, the filament structure is far from being resolved at the atomic level, which hampers rational understanding of IF pathologies. The elementary building block of all IF proteins is a dimer consisting of an α-helical coiled-coil (CC) “rod” domain flanked by the flexible head and tail domains. Here we present three crystal structures of overlapping human vimentin fragments that comprise the first half of its rod domain. Given the previously solved fragments, a nearly complete atomic structure of the vimentin rod has become available. It consists of three α-helical segments (coils 1A, 1B, and 2) interconnected by linkers (L1 and L12). Most of the CC structure has a left-handed twist with heptad repeats, but both coil 1B and coil 2 also exhibit untwisted, parallel stretches with hendecad repeats. In the crystal structure, linker L1 was found to be α-helical without being involved in the CC formation. The available data allow us to construct an atomic model of the antiparallel tetramer representing the second level of vimentin assembly. Although the presence of the nonhelical head domains is essential for proper tetramer stabilization, the precise alignment of the dimers forming the tetramer appears to depend on the complementarity of their surface charge distribution patterns, while the structural plasticity of linker L1 and coil 1A plays a role in the subsequent IF assembly process.

Chronic myeloid leukemia (CML) is a hematopoietic neoplasm characterized by the Philadelphia chromosome and the related BCR-ABL1 oncoprotein. Acceleration of CML is usually accompanied by basophilia. Several proangiogenic molecules have been implicated in disease acceleration, including the hepatocyte growth factor (HGF). However, little is known so far about the cellular distribution and function of HGF in CML. We here report that HGF is expressed abundantly in purified CML basophils and in the basophil-committed CML line KU812, whereas all other cell types examined expressed only trace amounts of HGF or no HGF. Interleukin 3, a major regulator of human basophils, was found to promote HGF expression in CML basophils. By contrast, BCR-ABL1 failed to induce HGF synthesis in CML cells, and imatinib failed to inhibit expression of HGF in these cells. Recombinant HGF as well as basophil-derived HGF induced endothelial cell migration in a scratch wound assay, and these effects of HGF were reverted by an anti-HGF antibody as well as by pharmacologic c-Met inhibitors. In addition, anti-HGF and c-Met inhibitors were found to suppress the spontaneous growth of KU812 cells, suggesting autocrine growth regulation. Together, HGF is a BCR-ABL1-independent angiogenic and autocrine growth regulator in CML. Basophils are a unique source of HGF in these patients and may play a more active role in disease-associated angiogenesis and disease progression than has so far been assumed. Our data also suggest that HGF and c-Met are potential therapeutic targets in CML.

The mechanical properties of epithelial cells are modulated by structural changes in keratin intermediate filament networks. To investigate the relationship between network architecture and viscoelasticity, we assembled keratin filaments from recombinant keratin proteins 8 (K8) and 18 (K18) in the presence of divalent ions (Mg2+). We probed the viscoelastic modulus of the network by tracking the movement of microspheres embedded in the network during assembly, and studied the network architecture using scanning electron microscopy. Addition of Mg2+ at physiological concentrations (

We have generated human recombinant keratins K8 and K18 and describe conditions to quantitatively follow their assembly into filaments. When renatured individually from 8M urea into a low ionic strength/high pH-buffer, K8 was present in a dimeric to tetrameric form as revealed by analytical ultracentrifugation. In contrast, K18 sedimented as a monomer. When mixed in 8 M urea and renatured together, K8 and K18 exhibited s-value profiles compatible with homogeneous tetrameric complexes. This finding was confirmed by sedimentation equilibrium centrifugation. Subsequently, these tetrameric starter units were subjected to assembly experiments at various protein concentrations. At low values such as 0.0025 g/l, unit-length filaments were abundantly present after 2s of assembly. During the following 5 min, filaments grew rapidly and by measuring the length of individual filaments we were able to generate time-dependent length profiles. These data revealed that keratins K8/K18 assemble several times faster than vimentin and desmin. In addition, we determined the persistence length l(p) of K8/K18 filaments to be in the range of 300 nm. Addition of 1 mM MgCl(2) increases l(p) to 480 nm indicating that magnesium ions affect the interaction of keratin subunits within the filament during assembly to some extent.

Heat shock protein 32 (Hsp32), also known as heme oxygenase 1 (HO-1), has recently been identified as a potential target in various hematologic malignancies. We provide evidence that Hsp32 is constitutively expressed in primary leukemic cells in patients with acute myeloid leukemia (AML) and in various AML cell lines (HL60, U937, KG1). Expression of Hsp32 mRNA was demonstrable by qPCR, and expression of the Hsp32 protein by immunocytochemistry and Western blotting. The stem cell-enriched CD34+/CD38+ and CD34+/CD38- fractions of AML cells were found to express Hsp32 mRNA in excess over normal CD34+ progenitor cells. Two Hsp32-targeting drugs, pegylated zinc-protoporphyrin (PEG-ZnPP) and styrene-maleic-acid-copolymer-micelle-encapsulated ZnPP (SMAZnPP), were found to inhibit cytokine-dependent and spontaneous proliferation in all 3 AML cell lines as well as in primary AML cells. Growth inhibitory effects of SMA-ZnPP and PEG-ZnPP were dose-dependent with IC50 values ranging between 1 and 20 μM, and were accompanied by apoptosis as evidenced by light- and electron microscopy, Tunel assay, and caspase-3 activation. Finally, we were able to demonstrate that SMA-ZnPP inhibits cytokine-dependent proliferation of CD34+/CD38+ and CD34+/CD38- AML progenitor cells in vitro in all patients as well as leukemiainitiation of AML stem cells in NOD-SCID IL-2Rγ(-/-) (NSG) mice in vivo. Together, our data suggest that Hsp32 plays an important role as a survival factor in leukemic stem cells and as a potential new target in AML.

The type III intermediate filaments (IFs) are essential cytoskeletal elements of mechanosignal transduction and serve critical roles in tissue repair. Mice genetically deficient for the IF protein vimentin (Vim(-/-)) have impaired wound healing from deficits in myofibroblast development. We report a surprising finding made in Vim(-/-) mice that corneas are protected from fibrosis and instead promote regenerative healing after traumatic alkali injury. This reparative phenotype in Vim(-/-) corneas is strikingly recapitulated by the pharmacological agent withaferin A (WFA), a small molecule that binds to vimentin and down-regulates its injury-induced expression. Attenuation of corneal fibrosis by WFA is mediated by down-regulation of ubiquitin-conjugating E3 ligase Skp2 and up-regulation of cyclin-dependent kinase inhibitors p27(Kip1) and p21(Cip1). In cell culture models, WFA exerts G(2)/M cell cycle arrest in a p27(Kip1)- and Skp2-dependent manner. Finally, by developing a highly sensitive imaging method to measure corneal opacity, we identify a novel role for desmin overexpression in corneal haze. We demonstrate that desmin down-regulation by WFA via targeting the conserved WFA-ligand binding site shared among type III IFs promotes further improvement of corneal transparency without affecting cyclin-dependent kinase inhibitor levels in Vim(-/-) mice. This dissociates a direct role for desmin in corneal cell proliferation. Taken together, our findings illuminate a previously unappreciated pathogenic role for type III IF overexpression in corneal fibrotic conditions and also validate WFA as a powerful drug lead toward anti-fibrosis therapeutic development.

Intermediate filament (IF) elongation proceeds via full-width “mini-filaments”, referred to as “unit-length” filaments (ULFs), which instantaneously form by lateral association of extended coiled-coil complexes after assembly is initiated. In a comparatively much slower process, ULFs longitudinally interact end-to-end with other ULFs to form short filaments, which further anneal with ULFs and with each other to increasingly longer filaments. This assembly concept was derived from time-lapse electron and atomic force microscopy data. We previously have quantitatively verified this concept through the generation of time-dependent filament length-profiles and an analytical model that describes assembly kinetics well for about the first ten minutes. In this time frame, filaments are shorter than one persistence length, i.e. ~1 μm, and thus filaments were treated as stiff rods associating via their ends. However, when filaments grow several μm in length over hours, their flexibility becomes a significant factor for the kinetics of the longitudinal annealing process. Incorporating now additional filament length distributions that we have recorded after extended assembly times by total internal reflection fluorescence microscopy (TIRFM), we developed a Monte Carlo simulation procedure that accurately describes the underlying assembly kinetics for large time scales.

Mutation of the LMNA gene, encoding nuclear lamin A and lamin C (hereafter lamin A/C), is a common cause of familial dilated cardiomyopathy (DCM). Among Finnish DCM patients, the founder mutation c.427T>C (p.S143P) is the most frequently reported genetic variant. Here, we show that p.S143P lamin A/C is more nucleoplasmic and soluble than wild-type lamin A/C and accumulates into large intranuclear aggregates in a fraction of cultured patient fibroblasts as well as in cells ectopically expressing either FLAG- or GFP-tagged p.S143P lamin A. In fluorescence loss in photobleaching (FLIP) experiments, non-aggregated EGFP-tagged p.S143P lamin A was significantly more dynamic. In in vitro association studies, p.S143P lamin A failed to form appropriate filament structures but instead assembled into disorganized aggregates similar to those observed in patient cell nuclei. A whole-genome expression analysis revealed an elevated unfolded protein response (UPR) in cells expressing p.S143P lamin A/C. Additional endoplasmic reticulum (ER) stress induced by tunicamycin reduced the viability of cells expressing mutant lamin further. In summary, p.S143P lamin A/C affects normal lamina structure and influences the cellular stress response, homeostasis and viability.

We have previously reported an increased expression of cytokeratins 8/18 (K8/K18) in cells expressing the F508del mutation of cystic fibrosis transmembrane conductance regulator (CFTR). This is associated with increased colocalization of CFTR and K18 in the vicinity of the endoplasmic reticulum, although this is reversed by treating cells with curcumin, resulting in the rescue of F508del-CFTR. In the present work, we hypothesized that (i) the K8/K18 network may interact physically with CFTR, and that (ii) this interaction may modify CFTR function. CFTR was immunoprecipitated from HeLa cells transfected with either wild-type (WT) CFTR or F508del-CFTR. Precipitates were subjected to 2D-gel electrophoresis and differential spots identified by mass spectrometry. K8 and K18 were found significantly increased in F508del-CFTR precipitates. Using surface plasmon resonance, we demonstrate that K8, but not K18, binds directly and preferentially to the F508del over the WT human NBD1 (nucleotide-binding domain-1). In vivo K8 interaction with F508del-CFTR was confirmed by proximity ligation assay in HeLa cells and in primary cultures of human respiratory epithelial cells. Ablation of K8 expression by siRNA in F508del-expressing HeLa cells led to the recovery of CFTR-dependent iodide efflux. Moreover, F508del-expressing mice topically treated with K8-siRNA showed restored nasal potential difference, equivalent to that of WT mice. These results show that disruption of F508del-CFTR and K8 interaction leads to the correction of the F508del-CFTR processing defect, suggesting a novel potential therapeutic target in CF.

The aim of this study is to determine when during hematopoiesis Siglec-8 gets expressed, whether it is expressed on hematologic malignancies, and if there are other non-human species that express Siglec-8.Siglec-8 mRNA and cell surface expression was monitored during in vitro maturation of human eosinophils and mast cells. Flow cytometry was performed on human blood and bone marrow samples, and on blood samples from dogs, baboons, and rhesus and cynomolgus monkeys.Siglec-8 is a late maturation marker. It is detectable on eosinophils and basophils from subjects with chronic eosinophilic leukemia, chronic myelogenous leukemia, and on malignant and non-malignant bone marrow mast cells, as well as the HMC-1.2 cell line. None of the Siglec-8 monoclonal antibodies tested recognized leukocytes from dogs, baboons, and rhesus and cynomolgus monkeys.Siglec-8-based therapies should not target immature human leukocytes but should recognize mature and malignant eosinophils, mast cells, and basophils. So far, there is no suitable species for preclinical testing of Siglec-8 monoclonal antibodies.

We have investigated and quantified the nuclear A-type lamin pool from human HeLa S3 suspension cells with respect to their distribution to detergent soluble and insoluble fractions. We devised a sequential extraction protocol and found that maximally 10% of A-type lamins are recovered in the soluble fraction. Notably, lamin C is enriched in low detergent fractions and only with 0.5% Nonidet P-40 lamin A and C are recovered in ratios nearly equivalent to those found in whole cell extracts and in the lamina fraction. Authentic nucleoplasmic proteins such as LAP2a, pRB and p53 are co-extracted to a large part together with the A-type lamins in these fractions. By sucrose density centrifugation we revealed that the majority of lamins co-sedimented with human IgG indicating they form rather small complexes in the range of dimers and slightly larger complexes. Some lamin A - but not lamin C - is obtained in addition in a much faster sedimenting fraction. Authentic nuclear proteins such as PCNA, p53 and LAP2a were found both in the light and the heavy sucrose fractions together with lamin A. Last but not least, immunoprecipitation experiments from both soluble fractions and from RIPA lysates of whole cells revealed that lamin A and lamin C do not form heterodimers but segregate practically completely. Correspondingly, immunofluorescence microscopy of formaldehyde-fixed cells clearly demonstrated that lamin A and C are localized at least in part to distinct patches within the lamina. Hence, the structural segregation of lamin A and C is indeed retained in the nuclear envelope to some extent too.

Epigenetic pathways can regulate gene expression by controlling and interpreting chromatin modifications. Cancer cells are characterized by altered epigenetic landscapes, and commonly exploit the chromatin regulatory machinery to enforce oncogenic gene expression programs1. Although chromatin alterations are, in principle, reversible and often amenable to drug intervention, the promise of targeting such pathways therapeutically has been limited by an incomplete understanding of cancer-specific dependencies on epigenetic regulators. Here we describe a non-biased approach to probe epigenetic vulnerabilities in acute myeloid leukaemia (AML), an aggressive haematopoietic malignancy that is often associated with aberrant chromatin states2. By screening a custom library of small hairpin RNAs (shRNAs) targeting known chromatin regulators in a genetically defined AML mouse model, we identify the protein bromodomain-containing 4 (Brd4) as being critically required for disease maintenance. Suppression of Brd4 using shRNAs or the small-molecule inhibitor JQ1 led to robust antileukaemic effects in vitro and in vivo, accompanied by terminal myeloid differentiation and elimination of leukaemia stem cells. Similar sensitivities were observed in a variety of human AML cell lines and primary patient samples, revealing that JQ1 has broad activity in diverse AML subtypes. The effects of Brd4 suppression are, at least in part, due to its role in sustaining Myc expression to promote aberrant self-renewal, which implicates JQ1 as a pharmacological means to suppress MYC in cancer. Our results establish small-molecule inhibition of Brd4 as a promising therapeutic strategy in AML and, potentially, other cancers, and highlight the utility of RNA interference (RNAi) screening for revealing epigenetic vulnerabilities that can be exploited for direct pharmacological intervention.

In acute lymphoblastic leukemia (ALL), the Philadelphia chromosome (Ph) is associated with a poor prognosis. For these patients, hematopoietic stem cell transplantation (HSCT) and BCR/ABL tyrosine kinase inhibitors (TKIs) are considered standard of therapy. However, it remains unclear whether BCR/ABL TKIs should be administered lifelong as maintenance post-HSCT, and whether the presence of minimal residual disease (MRD) is invariably associated with relapse. We report on two patients with Ph+ ALL who were successfully treated with polychemotherapy and consecutive autologous HSCT. Both patients are in continuous hematologic remission after an observation period of 12 years and 18 years, respectively, despite measurable MRD and although no maintenance therapy was initiated. BCR/ABL transcript-levels ranged between 0.1 and 3% in patient 1, and 0.01 and 0.1% in patient 2 during the observation time. Collectively, these data suggest that not all Ph+ subclones even those that persist after HSCT in Ph+ ALL, may have the potential to cause a hematologic relapse. We hypothesize that these small-sized clones are derived from neoplastic stem cells that have not (yet) accumulated a sufficient number of pro-oncogenic hits required for full transformation to ALL-initiating (stem) cells and thus overt leukemia.

The contribution of the intermediate filament (IF) network to the mechanical response of cells has so far received little attention, possibly because the assembly and regulation of IFs are not as well understood as that of the actin cytoskeleton or of microtubules. The mechanical role of IFs has been mostly inferred from measurements performed on individual filaments or gels in vitro. In this study we employ atomic force microscopy (AFM) to examine the contribution of vimentin IFs to the nanomechanical properties of living cells under native conditions. To specifically target and modulate the vimentin network, Rat-2 fibroblasts were transfected with GFP-desmin variants. Cells expressing desmin variants were identified by the fluorescence microscopy extension of the AFM instrument. This allowed us to directly compare the nanomechanical response of transfected and untransfected cells at high spatial resolution by means of AFM. Depending on the variant desmin, transfectants were either softer or stiffer than untransfected fibroblasts. Expression of the non-filament forming GFP-DesL345P mutant led to a collapse of the endogenous vimentin network in the perinuclear region that was accompanied by localized stiffening. Correlative confocal microscopy indicates that the expression of desmin variants specifically targets the endogenous vimentin IF network without major rearrangements of other cytoskeletal components. By measuring functional changes caused by IF rearrangements in intact cells, we show that IFs play a crucial role in mechanical behavior not only at large deformations but also in the nanomechanical response of individual cells.

The assembly of intermediate filaments (IFs) is a complex process that can be recapitulated through a series of distinct steps in vitro. The combination of microfluidics and small angle X-ray scattering (SAXS) provides a powerful tool to investigate the kinetics of this process on the relevant timescales. Microfluidic mixers based on the principle of hydrodynamic focusing allow for precise control of the mixing of proteins and smaller reagents like ions. Here, we present a multi-layer device that prevents proteins from adsorbing to the channel walls by engulfing the protein jet with a fluid layer of buffer. To ensure compatibility with SAXS, the device is fabricated from UV-curable adhesive (NOA 81). To demonstrate the successful prevention of contact between the protein jet and the channel walls we measure the distribution of a fluorescent dye in the device by confocal microscopy at various flow speeds and compare the results to finite element method (FEM) simulations. The prevention of contact enables the investigation of the assembly of IFs in flow by gradually increasing the salt concentration in the protein jet. The diffusion of salt into the jet can be determined by FEM simulations. SAXS data are collected at different positions in the jet, corresponding to different salt concentrations, and they reveal distinct differences between the earliest assembly states. We find that the mean square radius of gyration perpendicular to the filament axis increases from 13 nm(2) to 58 nm(2) upon assembly. Thereby we provide dynamic structural data of a complex assembly process that was amenable up to now only by microscopic techniques. peerReviewed

Neoplastic stem cells have initially been characterized in myeloid leukemias where NOD/SCID mouse-repopulating progenitors supposedly reside within a CD34+/Lin- subset of the malignant clone. These progenitors are considered to be self-renewing cells responsible for the in vivo long-term growth of neoplastic cells in leukemic patients. Therefore, these cells represent an attractive target of therapy. In some lymphoid leukemias, NOD/SCID mouse-repopulating cells were also reported to reside within the CD34+/Lin- subfraction of the clone. More recently, several attempts have been made to transfer the cancer stem cell concept to solid tumors and other non-hematopoietic neoplasms. In several of these tumors, the cell surface antigens AC133 (CD133) and CD44 are considered to indicate the potential of a cell to initiate permanent tumor formation in vivo. However, several questions concerning the phenotype, self-renewal capacity, stroma-dependence, and other properties of cancer- or leukemia-initiating cells remain to be solved. The current article provides a summary of our current knowledge on neoplastic (cancer) stem cells, with special emphasis on clinical implications and therapeutic options as well as a discussion about conceptual and technical limitations.

Waveguide circuits play a key role in modern integrated optics and provide an appealing approach to scalability in quantum optics. We report on periodically poled ridge waveguides in z-cut potassium titanyl phosphate (KTiOPO4 or KTP), a material that has recently received growing interest due to its unique dispersion properties. Ridges were defined in surface-near rubidium-exchanged KTP by use of a precise diamond-blade dicing saw. We fabricated single-mode ridge waveguides at around 800 nm which exhibit widths of 1.9–3.2 μm and facilitated type-II second harmonic generation from 792 nm to 396 nm with high efficiency of 6.6 %/W·cm2. Temperature dependence of the second harmonic process was found to be 53 pm/K. The low temperature dependence and high nonlinear conversion efficiency make our waveguides ideally suited for future operations in classical nonlinear integrated optics and integrated quantum networking applications.

Objective In most patients with systemic mastocytosis (SM), including aggressive SM (ASM) and mast cell (MC) leukemia (MCL), neoplastic cells express the oncogenic KIT mutation D816V, which confers resistance to imatinib. Cladribine (2CdA) is a nucleoside analog that has been introduced as a promising agent for treatment of advanced SM. Materials and Methods We examined the in vitro effects of 2CdA on growth of neoplastic MC, and the in vivo effects of 2CdA (0.13 mg/kg/day intravenously, days 1−5; three to eight cycles) in seven patients with advanced SM. Results Cladribine was found to inhibit growth of primary MC and the MC line HMC-1 in a dose-dependent manner, with lower IC 50 values recorded in HMC-1.2 cells harboring KIT D816V (IC 50 : 10 ng/mL) compared to HMC-1.1 cells lacking KIT D816V (IC 50 : 300 ng/mL). In two patients with progressive smoldering SM, 2CdA produced a long-lasting response with a sustained decrease in serum tryptase levels, whereas in patients with progressive ASM or MCL, 2CdA showed little if any effects. The drug was well-tolerated in most cases. However, one patient developed a massive generalized purulent long-lasting skin rash. The antiproliferative effects of 2CdA on MC were found to be associated with morphologic signs of apoptosis and caspase cleavage. Cladribine did not counteract the kinase activity of KIT D816V or KIT-downstream signaling molecules. Conclusions Cladribine may be a promising agent for treatment of progressive smoldering KIT D816V + SM. In rapidly progressing ASM or MCL, additional or alternative drugs are required to induce long-lasting antineoplastic effects.

The nuclear envelope (NE) is a barrier that separates nuclear from cytoplasmic processes. It is composed of an inner and outer nuclear membrane (INM, ONM), separated by the perinuclear space (PNS). The ONM is contiguous with the endoplasmic reticulum (ER), and thus, the lumen of the NE and that of the ER constitute one compartment. The lamin B receptor (LBR) is a NE protein that has a central structural role as a linker of the INM, the lamina and chromatin, and a less well characterized functional role as a sterol reductase. In a recent study, we reported that the forced expression of mutant variants of LBR in some cell types induces a separation of the INM from the outer nuclear envelope concomitantly with a separation of ER membranes, whereas in other cells no separation is observed. In this extra view, we speculate about the mechanism that leads to this fundamental disruption of NE and ER structure. Our observations furthermore raise the question to what extent LBR contributes to the establishment or maintenance of the ER and PNS luminal compartment, and how a single mutant protein can so drastically interfere with its regular organization.

Seit vielen Jahren wird die Biologie der Tumorzellen und ihre Bedeutung fur Tumorprogression, Metastasierung und Prognose der Tumorpatienten erforscht. In den letzten Jahren gewinnt dabei das Konzept der sogenannten Tumor-Stammzellen immer mehr an Bedeutung. Dieses Modell basiert auf der Beobachtung, dass das kontinuierliche Wachstum von Tumoren und Leukamien von einer kleinen Population sehr unreifer neoplastischer Zellen, den Tumorstammzellen abhangt, wahrend die reiferen Zellen der Neoplasie nach einer variablen Anzahl von Zellteilungen uber Apoptose absterben. Die Selbsterneuerungsfahigkeit der Tumorstammzellen spielt dabei eine zentrale Rolle und ermoglicht eine dauerhafte Repopulation in vivo im Patienten und in experimentellen Modellen wie z.B. in immunsupprimierten Mausen. Somit ist auch klar, dass antineoplastische Therapien nur dann ein kuratives Potential haben, wenn die Tumorstammzellen getroffen werden. Ein wichtiger Aspekt ist deren intrinsische Resistenz gegenuber konventionellen Medikamenten. Daher versucht man, molekulare Targets und Target-Expressionsprofile in neoplastischen Stammzellen zu erkennen und in therapeutischen Ansatzen zu nutzen. Es ist zu erhoffen, dass die Anwendung der Tumorstammzell-Konzepte zu einer nachhaltigen Verbesserung der Therapie von Leukamien und Tumorerkrankungen fuhren wird.

Inherited mutations in the gene coding for the intermediate filament protein desmin have been demonstrated to cause severe skeletal and cardiac myopathies. Unexpectedly, some of the mutated desmins, in particular those carrying single amino acid alterations in the non-alpha-helical carboxy-terminal domain ("tail"), have been demonstrated to form apparently normal filaments both in vitro and in transfected cells. Thus, it is not clear if filament properties are affected by these mutations at all. For this reason, we performed oscillatory shear experiments with six different desmin "tail" mutants in order to characterize the mesh size of filament networks and their strain stiffening properties. Moreover, we have carried out high-frequency oscillatory squeeze flow measurements to determine the bending stiffness of the respective filaments, characterized by the persistence length l(p). Interestingly, mesh size was not altered for the mutant filament networks, except for the mutant DesR454W, which apparently did not form proper filament networks. Also, the values for bending stiffness were in the same range for both the "tail" mutants (l(p)=1.0-2.0 microm) and the wild-type desmin (l(p)=1.1+/-0.5 microm). However, most investigated desmin mutants exhibited a distinct reduction in strain stiffening compared to wild-type desmin and promoted nonaffine network deformation. Therefore, we conclude that the mutated amino acids affect intrafilamentous architecture and colloidal interactions along the filament in such a way that the response to applied strain is significantly altered. In order to explore the importance of the "tail" domain as such for filament network properties, we employed a "tail"-truncated desmin. Under standard conditions, it formed extended regular filaments, but failed to generate strain stiffening. Hence, these data strongly indicate that the "tail" domain is responsible for attractive filament-filament interactions. Moreover, these types of interactions may also be relevant to the network properties of the desmin cytoskeleton in patient muscle.

In this paper, we report on an integrated optical wavelength filter device in LiNbO3 . A sequence of individually addressable polarization converters enables programmable spectral response and continuous wavelength tunability. Wavelength-selective polarization conversion is obtained using in-phase and quadrature-driven electro-optic converters. A model based on coupled-mode analysis is presented that allows to determine the filter performance. Examples of tailoring the spectral response to obtain strong sidelobe suppression and flat-top responses are discussed. Predicted theoretical results are confirmed by experiments. A 2.3 nm wide bandpass filter with more than 20 dB sidelobe suppression and a tuning range of about 20 nm are demonstrated. Moreover, even a flat-top characteristics with 4.9 nm bandwidth and about 15 dB sidelobe suppression could be demonstrated with the same device.

Chronic myeloid leukemia (CML) is a stem cell (SC) neoplasm characterized by the BCR/ABL1 oncogene. Although the disease can be kept under control using BCR/ABL1 tyrosine kinase inhibitors (TKIs) in most cases, some patients relapse or have resistant disease, so there is a need to identify new therapeutic targets in this malignancy. Recent data suggest that leukemic SCs (LSCs) in CML display the stem-cell (SC)-mobilizing cell surface enzyme dipeptidyl-peptidase IV (DPPIV = CD26) in an aberrant manner. In the present study, we analyzed the effects of the DPPIV blocker vildagliptin as single agent or in combination with the BCR/ABL1 TKI imatinib or nilotinib on growth and survival of CML LSCs in vitro and on LSC engraftment in an in vivo xenotransplantation nonobese diabetic SCID-IL-2Rγ(−/−) (NSG) mouse model. We found that nilotinib induces apoptosis in CML LSCs and inhibits their engraftment in NSG mice. In contrast, no substantial effects were seen with imatinib or vildagliptin. Nevertheless, vildagliptin was found to reduce the “mobilization” of CML LSCs from a stroma cell layer consisting of mouse fibroblasts in an in vitro co-culture model, suggesting reduced disease expansion. However, although vildagliptin and nilotinib produced cooperative effects in individual experiments, overall, no significant effects of coadministered vildagliptin over nilotinib or imatinib treatment alone were seen on the engraftment of CML cells in NSG mice. Gliptins may be interesting drugs in the context of CML and nilotinib therapy, but our preclinical studies did not reveal a major cooperative effect of the drug-combination vildagliptin + nilotinib on engraftment of CML cells in NSG mice.

We report on the fabrication and characterization of ridge waveguides in z-cut KTiOPO4 fabricated by Rb-ion exchange and subsequent precise diamond-blade dicing. Low attenuation values of 1.3 dB/cm (1.6 dB/cm) were determined at a wavelength of 1550 nm for TE (TM) polarization. Surface quality obtained by dicing is excellent for side walls of diced ridges and prepared end faces used for light coupling. The dispersion characteristics of the waveguides are determined and the results are compared to simulations. Finally, the nonlinear performance of the ridges is demonstrated by second harmonic generation of ∼1060 nm pump light.

Numerous mutations in the human A-type lamin gene (LMNA) cause the premature aging disease, progeria. Some of these are located in the α-helical central rod domain required for the polymerization of the nuclear lamins into higher order structures. Patient cells with a mutation in this domain, 433G>A (E145K) show severely lobulated nuclei, a separation of the A- and B-type lamins, alterations in pericentric heterochromatin, abnormally clustered centromeres, and mislocalized telomeres. The induction of lobulations and the clustering of centromeres originate during postmitotic nuclear assembly in daughter cells and this early G1 configuration of chromosomes is retained throughout interphase. In vitro analyses of E145K-lamin A show severe defects in the assembly of protofilaments into higher order lamin structures. The results show that this central rod domain mutation affects nuclear architecture in a fashion distinctly different from the changes found in the most common form of progeria caused by the expression of LAΔ50/progerin. The study also emphasizes the importance of lamins in nuclear assembly and chromatin organization.