An improved proton NMR method for the real‐time measurement of the hard/soft ratio or the crystallinity, and the mobile‐fraction dynamics, in phase‐separated or semicrystalline polymers is presented. ...It avoids some difficulties associated with earlier approaches and can be applied on high‐ as well as inexpensive low‐field instrumentation. A pulsed mixed magic‐sandwich echo is shown to provide near‐quantitative refocusing of the rigid contribution to the initial part of the free induction decay. This essentially removes the need to account for signal loss during the receiver dead time, and the method should thus be useful for a variety of applications where the magnetization distribution over differently mobile fractions is to be determined. The overall decay of the mobile signal of a semicrystalline polymer was found to exhibit a significant field dependence, such that the apparent transverse relaxation function of the amorphous part is in a real‐time experiment best characterized by a subsequent Carr–Purcell–Meiboom–Gill pulse train. It is demonstrated to be mainly influenced by mobility, while instrumental effects play a minor role. The mobility of the amorphous fraction depends not only on the overall crystallinity, but also on the crystallization conditions, thus on the nanometer‐scale morphology.
Isothermal crystallization of sPP monitored on a 20 MHz low‐field proton NMR spectrometer using the MSE‐CPMG experiment.
Glioblastoma multiforme (GBM), or grade IV astrocytoma, is the most common type of primary brain tumor. It has a devastating prognosis with a 2-year-overall survival rate of only 26 % after standard ...treatment, which includes surgery, radiation, and adjuvant chemotherapy with temozolomide. Also lower grade gliomas are difficult to treat, because they diffusely spread into the brain, where extensive removal of tissue is critical. Better understanding of the cancer’s biology is a key for the development of more effective therapy approaches. The discovery of isocitrate dehydrogenase (IDH) mutations in leukemia and glioma drew attention to specific metabolic aberrations in IDH-mutant gliomas. In the center of the metabolic alterations is α-ketoglutarate (αKG), an intermediate metabolite in the tricarboxylic acid (TCA) cycle, and the associated amino acid glutamate (Glu). This article highlights the role of these metabolites in glioma energy and lipid production and indicates possible weak spots of IDH-mutant and IDH-wt gliomas.
Induced pluripotent stem cells (iPSCs) provide a unique opportunity for generation of patient-specific cells for use in translational purposes. We aimed to compare iPSCs generated by different ...reprogramming methods regarding their reprogramming efficiency, pluripotency capacity, and the possibility to use high-throughput PCR-based methods for detection of human pathogenic viruses. iPSCs from skin fibroblasts (FB), peripheral blood mononuclear cells (PBMCs), or mesenchymal stem cells (MSCs) were generated by using three different reprogramming systems including chromosomal integrating and nonintegrating methods. Reprogramming efficiencies were in accordance with the literature, indicating that the parental cell type and the reprogramming method play a major role for the reprogramming efficiencies (FB: STEMCCA: 1.30±0.18, Sendai virus: 1.37±0.01, and episomal plasmids: 0.04±0.02; PBMCs: Sendai virus: 0.002±0.001, episomal plasmids: 0) but result in the same characteristics of pluripotency. We found the highest reprogramming efficiencies for MSC with 3.32±1.2 by using episomal plasmids. Since GMP standard working procedures and screening units need virus contamination-free cell lines, we studied HIV-1 contamination in the generated iPSCs. We used the high-throughput cobas® 6800/8800 system, which is normally used for detection of HIV-1 in plasma of patients, and found that footprint-free reprogramming methods as episomal plasmids and Sendai virus are useful for the described virus detection method. This fast, cost-effective, robust, and reliable assay demonstrates the feasibility to use high-throughput PCR-based methods for detection of human pathogenic viruses in ps-iPSC lines that were generated with nongenome integrating reprogramming methods.
Doxorubicin (DOX) has been used for decades to treat hematopoietic and solid tumors, although, in a subset of cancer survivors,it causes cardiotoxicity decades after treatment. The mechanisms of ...anthracycline-induced cardiotoxicity (ACT) are still incompletely understood. Induced pluripotent stem cells (iPSCs)-derived cardiomyocytes have become a valuable tool to study hereditary and structural cardiac conditions in vitro. A former study from our group showed that iPSC-derived cardiomyocytes (iPSC-CMs) from lymphoma patients with ACT were continuously more sensitive to DOX toxicity,demonstrating disorganized myofilament structure and altered calcium handling along with increased cell death and reactive oxygen species (ROS) production compared to control iPSC-CMs. The aims of this study are to (1) analyze the cellular resorption and distribution of DOX, (2) assess itsimpact on mRNA translation, (3) address an ACT-associated single-nucleotide polymorphism (SNP) (rs13058338 in the RAC2 gene) in ACT disease progression, (4) test a potential treatment option with TERT overexpression, and finally, (5) distinguish between ACT and unspecific effects of DOX in human cardiomyocytes. To this end, iPSCs from ACT-and dilated cardiomyopathy (DCM)-patients as well as control iPSC lines were differentiated into three-month-old iPSC-CMs and treated with DOX to model the effects of the drug exposure on human cardiomyocytes. In the first part of the study, the cellular resorption and retention of DOX in iPSC-CMs was investigated using high-performance liquid chromatography (HPLC) and positive correlations between DOX treatment time, treatment concentration and intracellular DOX levels were found. Intracellular DOX levels were consistently higher in ACT-iPSC-CMs compared to control iPSC-CMs and DOX remained longer in ACT-iPSC-CMs after treatment compared to control iPSC-CMs.
Cancer therapies with anthracyclines have been shown to induce cardiovascular complications. The aims of this study were to establish an in vitro induced pluripotent stem cell model (iPSC) of ...anthracycline-induced cardiotoxicity (ACT) from patients with an aggressive form of B-cell lymphoma and to examine whether doxorubicin (DOX)-treated ACT-iPSC cardiomyocytes (CM) can recapitulate the clinical features exhibited by patients, and thus help uncover a DOX-dependent pathomechanism. ACT-iPSC CM generated from individuals with CD20
+
B-cell lymphoma who had received high doses of DOX and suffered cardiac dysfunction were studied and compared to control-iPSC CM from cancer survivors without cardiac symptoms. In cellular studies, ACT-iPSC CM were persistently more susceptible to DOX toxicity including augmented disorganized myofilament structure, changed mitochondrial shape, and increased apoptotic events. Consistently, ACT-iPSC CM and cardiac fibroblasts isolated from fibrotic human ACT myocardium exhibited higher DOX-dependent reactive oxygen species. In functional studies, Ca
2+
transient amplitude of ACT-iPSC CM was reduced compared to control cells, and diastolic sarcoplasmic reticulum Ca
2+
leak was DOX-dependently increased. This could be explained by overactive CaMKIIδ in ACT CM. Together with DOX-dependent augmented proarrhythmic cellular triggers and prolonged action potentials in ACT CM, this suggests a cellular link to arrhythmogenic events and contractile dysfunction especially found in ACT engineered human myocardium. CamKIIδ inhibition prevented proarrhythmic triggers in ACT. In contrast, control CM upregulated SERCA2a expression in a DOX-dependent manner, possibly to avoid heart failure conditions. In conclusion, we developed the first human patient-specific stem cell model of DOX-induced cardiac dysfunction from patients with B-cell lymphoma. Our results suggest that DOX-induced stress resulted in arrhythmogenic events associated with contractile dysfunction and finally in heart failure after persistent stress activation in ACT patients.
The malignant primary brain tumor, glioblastoma (GBM) is generally incurable. New approaches are desperately needed. Adeno-associated virus (AAV) vector-mediated delivery of anti-tumor transgenes is ...a promising strategy, however direct injection leads to focal transgene spread in tumor and rapid tumor division dilutes out the extra-chromosomal AAV genome, limiting duration of transgene expression. Intravenous (IV) injection gives widespread distribution of AAV in normal brain, however poor transgene expression in tumor, and high expression in non-target cells which may lead to ineffective therapy and high toxicity, respectively. Delivery of transgenes encoding secreted, anti-tumor proteins to tumor stromal cells may provide a more stable and localized reservoir of therapy as they are more differentiated than fast-dividing tumor cells. Reactive astrocytes and tumor-associated macrophage/microglia (TAMs) are stromal cells that comprise a large portion of the tumor mass and are associated with tumorigenesis. In mouse models of GBM, we used IV delivery of exosome-associated AAV vectors driving green fluorescent protein expression by specific promoters (NF-κB-responsive promoter and a truncated glial fibrillary acidic protein promoter), to obtain targeted transduction of TAMs and reactive astrocytes, respectively, while avoiding transgene expression in the periphery. We used our approach to express the potent, yet toxic anti-tumor cytokine, interferon beta, in tumor stroma of a mouse model of GBM, and achieved a modest, yet significant enhancement in survival compared to controls. Noninvasive genetic modification of tumor microenvironment represents a promising approach for therapy against cancers. Additionally, the vectors described here may facilitate basic research in the study of tumor stromal cells in situ.
Graphical abstract
•PDAC and colon cancer (CRC) cell lines constitutively express HIF-1α.•HIF-1α inhibition enhances cell surface α1,2-fucosylation on PDAC and CRC cells.•Inhibition of HIF-1α enhances FUT1 and FUT2 ...transcripts in PDAC and CRC cells.•Knock down of HIF-1α increases FUT1/2 expression and α1,2-fucosylation in PaTu-T.•HIF-1α suppresses FUT1/2 expression, thus regulating cell surface α1,2-fucosylation.
The α1,2-fucosyltransferase activity in pancreatic tumors is much lower compared to normal pancreatic tissue. Here we show that hypoxia inducible factor (HIF) 1α is constitutively expressed in the pancreatic cancer cell lines Pa-Tu-8988S and Pa-Tu-8988T and suppresses the expression of the α1,2-fucosyltransferase genes FUT1 and FUT2. Down regulation of HIF-1α expression resulted in elevated FUT1 and FUT2 transcript levels and an increased expression of α1,2-fucosylated glycan structures on the surface of these cells. In conclusion, our data are the first to identify HIF-1α as a suppressor of FUT1/2 expression, thereby regulating α1,2-fucosylation of cell-surface glycans.
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