Multislice localized parallel excitation for EPI applications in humans

In this work, the opportunities and challenges for the use of parallel transmission in combination with 2D RF pulses designed on EPI‐based excitation trajectories for diffusion‐weighted imaging (DWI) with reduced FOV are presented and analyzed in detail. The use of localized excitation allows for shortening of the EPI read‐out, which is especially important for EPI applications outside of the brain. DWI is chosen as a practically important and relevant example demonstrating the key aspects of 2D spatial selection. The properties of accelerated pulses are explored experimentally in phantoms for two different schemes, in which the thickness of the excited limited slices is encoded either along the frequency or phase encoding directions of the excitation trajectory. The feasibility of application of parallel transmission for MR imaging in humans is analyzed based on several pilot experiments. Although the parallel transmission acceleration is demonstrated to work in some examples in the spinal cord and abdomen, the results also uncover a number of challenges. Nonetheless, the reduction of FOV in the phase encoding direction of the read‐out train along with the associated substantial shortening of the minimum echo train length and reduction of geometric distortions motivates further search for an advantageous use of the parallel transmit technology in EPI applications. © 2015 Wiley Periodicals, Inc. Concepts Magn Reson Part B (Magn Reson Engineering) 45B: 153–173, 2015     

Loss of diphthamide pre-activates NF-κB and death receptor pathways and renders MCF7 cells hypersensitive to tumor necrosis factor

The diphthamide on human eukaryotic translation elongation factor 2 (eEF2) is the target of ADP ribosylating diphtheria toxin (DT) and Pseudomonas exotoxin A (PE). This modification is synthesized by seven dipthamide biosynthesis proteins (DPH1–DPH7) and is conserved among eukaryotes and archaea. We generated MCF7 breast cancer cell line-derived DPH gene knockout (ko) cells to assess the impact of complete or partial inactivation on diphthamide synthesis and toxin sensitivity, and to address the biological consequence of diphthamide deficiency. Cells with heterozygous gene inactivation still contained predominantly diphthamide-modified eEF2 and were as sensitive to PE and DT as parent cells. Thus, DPH gene copy number reduction does not affect overall diphthamide synthesis and toxin sensitivity. Complete inactivation of DPH1, DPH2, DPH4, and DPH5 generated viable cells without diphthamide. DPH1ko, DPH2ko, and DPH4ko harbored unmodified eEF2 and DPH5ko ACP- (diphthine-precursor) modified eEF2. Loss of diphthamide prevented ADP ribosylation of eEF2, rendered cells resistant to PE and DT, but does not affect sensitivity toward other protein synthesis inhibitors, such as saporin or cycloheximide. Surprisingly, cells without diphthamide (independent of which the DPH gene compromised) were presensitized toward nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) and death-receptor pathways without crossing lethal thresholds. In consequence, loss of diphthamide rendered cells hypersensitive toward TNF-mediated apoptosis. This finding suggests a role of diphthamide in modulating NF-κB, death receptor, or apoptosis pathways.Eukaryotic translation elongation factor 2 (eEF2) is a highly conserved protein and essential for protein biosynthesis. EEF2 enables peptide-chain elongation by translocating the peptide–tRNA complex from the A- to the P-site of the ribosome (1, 2). The diphthamide modification at His715 of human eEF2 (or at the corresponding position in other species) is conserved in all eukaryotes (3) and in archaeal counterparts. It is generated by proteins that are encoded by seven genes (4). Proteins encoded by dipthamide biosynthesis protein (DPH)1, DPH2, DPH3, and DPH4 (DNAJC24) attach a 3-amino-3-carboxypropyl (ACP) group to eEF2. This intermediate is converted by the methyltransferase DPH5 to diphthine, which is subsequently amidated to diphthamide by DPH6 and DPH7 (5).Diphthamide synthesis was previously described in yeast and other eukaryotes (4–6). However, the “complete picture” is (with the exception of the yeast pathway) to a large portion is composed of observations made in different cell types on single genes. Many reports related to diphthamide synthesis of mammalian cells describe “partial knockouts” and “partial phenotypes” (i.e., reduced levels but not complete loss of diphthamide modification or toxin sensitivities) (7–9). Because mammalian genomes are more complex than that of yeast, carrying extendend gene families, mammalian cells may compensate—at least to some degree—functional loss of genes that may be unique and essential in yeast. If and to what degree mammalian cells can compensate a partial or complete loss of DPH gene functionality (and with what consequences) is unknown to date.So far, the function of diphthamide on eEF2 also remained rather elusive. Reports indicate that it contributes to translation fidelity (10–13). On the other hand, DPH genes or eEF2 can be mutated to prevent diphthamide attachment, yet cells carrying such mutations are viable (5, 11, 14, 15). Animals with heterozygous DPH knockouts (DPHko) can be generated, but homozygous DPH1ko, DPH3ko, and DPH4ko are embryonic lethal (13, 16–18). Because these studies are based on inactivation of individual genes, it is difficult to discriminate between phenotypes caused by gene loss and phenotypes as a consequence of loss of diphthamide.Diphthamide-modified eEF2 is the target of ADP ribosylating toxins, including Pseudomonas exotoxin A (PE) and diphtheria toxin (DT) (19). These bacterial proteins enter cells and catalyze ADP ribosylation of diphthamide using nictotinamide adenine dinucleotide (NAD) as substrate (20, 21). This inactivates eEF2, arrests protein synthesis, and kills (14). Tumor-targeted PE and DT derivatives are applied in cancer therapies (22–28) and their efficacy depends on toxin sensitivity of target cells. Therefore, information about factors (and their relative contributions) that influences cellular sensitivities toward diphthamide-modifying toxins may predict therapy responses. For example, alterations in OVCA1 (human DPH1) were described for ovarian cancers (16, 29), yet it is not known if and to what degree such alterations would affect sensitivities of tumor cells toward PE-derived drugs.Here we describe MCF7 breast cancer cell line derivatives with heterozygous or complete DPH gene inactivations. These cells are applied to analyze the contributions of individual DPHs not only to diphthamide synthesis and toxin sensitivity, but also to address gene dose effects. Because the set of knockout cell lines is derived from the same parent cell and provides loss of diphthamide as common consequence of inactivation of different genes, these cells can also shed light on the biological relevance of the diphthamide modification.     

Potential therapeutic target for malignant paragangliomas: ATP synthase on the surface of paraganglioma cells

F1FoATP synthase (ATP synthase) is a ubiquitous enzyme complex in eukaryotes. In general it is localized to the mitochondrial inner membrane and serves as the last step in the mitochondrial oxidative phosphorylation of ADP to ATP, utilizing a proton gradient across the inner mitochondrial membrane built by the complexes of the electron transfer chain. However some cell types, including tumors, carry ATP synthase on the cell surface. It was suggested that cell surface ATP synthase helps tumor cells thriving on glycolysis to survive their high acid generation. Angiostatin, aurovertin, resveratrol, and antibodies against the α and β subunits of ATP synthase were shown to bind and selectively inhibit cell surface ATP synthase, promoting tumor cell death. Here we show that ATP synthase β (ATP5B) is present on the cell surface of mouse pheochromocytoma cells as well as tumor cells of human SDHB-derived paragangliomas (PGLs), while being virtually absent on chromaffin primary cells from bovine adrenal medulla by confocal microscopy. The cell surface location of ATP5B was verified in the tissue of an SDHB-derived PGL by immunoelectron microscopy. Treatment of mouse pheochromocytoma cells with resveratrol as well as ATP5B antibody led to statistically significant proliferation inhibition. Our data suggest that PGLs carry ATP synthase on their surface that promotes cell survival or proliferation. Thus, cell surface ATP synthase may present a novel therapeutic target in treating metastatic or inoperable PGLs.     

Alveolar corticotomies for accelerated orthodontics: A systematic review

Introduction

It has been suggested that alveolar corticotomies may accelerate tooth movement, broaden the scope of malocclusion types that can be treated orthodontically, decrease the need for extractions, and support long-term stability. Several techniques have been proposed, although the indications, ideal design and technical characteristics, potential complications, and objective clinician and patient satisfaction remain unclear. This systematic review aimed to provide scientific support to validate alveolar corticotomies as a reliable approach to accelerated orthodontics.