The ND2 subunit is labeled by a photoaffinity analogue of asimicin, a potent complex I inhibitor

AbstractNADH:ubiquinone oxidoreductase (complex I) is the entry enzyme of mitochondrial oxidative phosphorylation. To obtain the structural information on inhibitor/quinone binding sites, we synthesized [3H]benzophenone-asimicin ([3H]BPA), a photoaffinity analogue of asimicin, which belongs to the acetogenin family known as the most potent complex I inhibitor. We found that [3H]BPA was photo-crosslinked to ND2, ND1 and ND5 subunits, by the three dimensional separation (blue-native/doubled SDS–PAGE) of [3H]BPA-treated bovine heart submitochondrial particles. The cross-linking was blocked by rotenone. This is the first finding that ND2 was photo-crosslinked with a potent complex I inhibitor, suggesting its involvement in the inhibitor/quinone-binding

Development of a spectro-electrochemical cell for soft X-ray photon-in photon-out spectroscopy

We developed a spectro-electrochemical cell for X-ray absorption and X-ray emission spectroscopy, which are element-specific methods to study local electronic structures in the soft X-ray region. In the usual electrochemical measurement setup, the electrode is placed in solution, and the surface/interface region of the electrode is not normally accessible by soft X-rays that have low penetration depth in liquids. To realize soft X-ray observation of electrochemical reactions, a 15-nm-thick Pt layer was deposited on a 150-nm-thick film window with an adhesive 3-nm-thick Ti layer for use as both the working electrode and the separator window between vacuum and a sample liquid under atmospheric pressure. The designed three-electrode electrochemical cell consists of a Pt film on a SiC window, a platinized Pt wire, and a commercial Ag|AgCl electrode as the working, counter, and reference electrodes, respectively. The functionality of the cell was tested by cyclic voltammetry and X-ray absorption and emission spectroscopy. As a demonstration, the electroplating of Pb on the Pt/SiC membrane window was measured by X-ray absorption and real-time monitoring of fluorescence intensity at the O 1s excitation

Categorization of 77 dystrophin exons into 5 groups by a decision tree using indexes of splicing regulatory factors as decision markers

<p>Abstract</p> <p>Background</p> <p>Duchenne muscular dystrophy, a fatal muscle-wasting disease, is characterized by dystrophin deficiency caused by mutations in the <it>dystrophin </it>gene. Skipping of a target <it>dystrophin </it>exon during splicing with antisense oligonucleotides is attracting much attention as the most plausible way to express dystrophin in DMD. Antisense oligonucleotides have been designed against splicing regulatory sequences such as splicing enhancer sequences of target exons. Recently, we reported that a chemical kinase inhibitor specifically enhances the skipping of mutated <it>dystrophin </it>exon 31, indicating the existence of exon-specific splicing regulatory systems. However, the basis for such individual regulatory systems is largely unknown. Here, we categorized the <it>dystrophin </it>exons in terms of their splicing regulatory factors.</p> <p>Results</p> <p>Using a computer-based machine learning system, we first constructed a decision tree separating 77 authentic from 14 known cryptic exons using 25 indexes of splicing regulatory factors as decision markers. We evaluated the classification accuracy of a novel cryptic exon (exon 11a) identified in this study. However, the tree mislabeled exon 11a as a true exon. Therefore, we re-constructed the decision tree to separate all 15 cryptic exons. The revised decision tree categorized the 77 authentic exons into five groups. Furthermore, all nine disease-associated novel exons were successfully categorized as exons, validating the decision tree. One group, consisting of 30 exons, was characterized by a high density of exonic splicing enhancer sequences. This suggests that AOs targeting splicing enhancer sequences would efficiently induce skipping of exons belonging to this group.</p> <p>Conclusions</p> <p>The decision tree categorized the 77 authentic exons into five groups. Our classification may help to establish the strategy for exon skipping therapy for Duchenne muscular dystrophy.</p

Long-term activation of anti-tumor immunity in pancreatic cancer by a p53-expressing telomerase-specific oncolytic adenovirus

Background: Pancreatic cancer is an aggressive, immunologically “cold” tumor. Oncolytic virotherapy is a promising treatment to overcome this problem. We developed a telomerase-specific oncolytic adenovirus armed with p53 gene (OBP-702). Methods: We investigated the efficacy of OBP-702 for pancreatic cancer, focusing on its long-term effects via long-lived memory CD8 + T cells including tissue-resident memory T cells (TRMs) and effector memory T cells (TEMs) differentiated from effector memory precursor cells (TEMps). Results: First, in vitro, OBP-702 significantly induced adenosine triphosphate (ATP), which is important for memory T cell establishment. Next, in vivo, OBP-702 local treatment to murine pancreatic PAN02 tumors increased TEMps via ATP induction from tumors and IL-15Rα induction from macrophages, leading to TRM and TEM induction. Activation of these memory T cells by OBP-702 was also maintained in combination with gemcitabine+nab-paclitaxel (GN) in a PAN02 bilateral tumor model, and GN + OBP-702 showed significant anti-tumor effects and increased TRMs in OBP-702-uninjected tumors. Finally, in a neoadjuvant model, in which PAN02 cells were re-inoculated after resection of treated-PAN02 tumors, GN + OBP-702 provided long-term anti-tumor effects even after tumor resection. Conclusion: OBP-702 can be a long-term immunostimulant with sustained anti-tumor effects on immunologically cold pancreatic cancer

Local oncolytic adenovirotherapy produces an abscopal effect via tumor-derived extracellular vesicles

Extracellular vesicles (EVs) play important roles in various intercellular communication processes. The abscopal effect is an interesting phenomenon in cancer treatment, in which immune activation is generally considered a main factor. We previously developed a telomerase-specific oncolytic adenovirus, Telomelysin (OBP-301), and occasionally observed therapeutic effects on distal tumors after local treatment in immunodeficient mice. In this study, we hypothesized that EVs may be involved in the abscopal effect of OBP-301. EVs isolated from the supernatant of HCT116 human colon carcinoma cells treated with OBP-301 were confirmed to contain OBP-301, and they showed cytotoxic activity (apoptosis and autophagy) similar to OBP-301. In bilateral subcutaneous HCT116 and CT26 tumor models, intratumoral administration of OBP-301 produced potent antitumor effects on tumors that were not directly treated with OBP-301, involving direct mediation by tumor-derived EVs containing OBP-301. This indicates that immune activation is not the main factor in this abscopal effect. Moreover, tumor-derived EVs exhibited high tumor tropism in orthotopic HCT116 rectal tumors, in which adenovirus E1A and adenovirus type 5 proteins were observed in metastatic liver tumors after localized rectal tumor treatment. In conclusion, local treatment with OBP-301 has the potential to produce abscopal effects via tumor-derived EVs

Transforming Growth Factor-β Induces Interleukin-6 Secretion from Human Ligamentum Flavum–Derived Cells through Partial Activation of p38 and p44/42 Mitogen-Activated Protein Kinases

Study Design This experimental study was performed using human ligamentum flavum–derived cells (HFCs). Purpose To investigate the intracellular signaling mechanism of interleukin-6 (IL-6) secretion in transforming growth factor-β (TGF-β)-stimulated HFCs. Overview of Literature Lumbar spinal stenosis (LSS) is a prevalent disease among the elderly, characterized by debilitating pain in the lower extremities. Although the number of patients with LSS has increased in recent years, the underlying pathomechanism remains unclear. Clinical examinations typically rely on magnetic resonance imaging to diagnose patients, revealing ligamentum flavum hypertrophy. Some studies have suggested an association between ligamentum flavum hypertrophy and inflammation/fibrosis, and expression of TGF-β and IL-6 has been observed in surgically obtained ligamentum flavum samples. However, direct evidence linking TGF-β and IL-6 expression in HFCs is lacking. Methods HFCs were obtained from patients with LSS who had undergone decompression surgery. The cells were stimulated with TGF-β and pretreated with either the p38 mitogen-activated protein (MAP) kinase inhibitor SB203580 or the p44/42 MAP kinase inhibitor FR180204. IL-6 secretion in the cell culture medium and IL-6 messenger RNA (mRNA) expression levels were analyzed using an enzyme-linked immunoassay and real-time polymerase chain reaction, respectively. Results TGF-β administration resulted in a dose- and time-dependent stimulation of IL-6 release. Treatment with SB203580 and FR180204 markedly suppressed TGF-β–induced IL-6 secretion from HFCs. Moreover, these inhibitors suppressed IL-6 mRNA expression in response to TGF-β stimulation. Conclusions Our findings indicate that TGF-β induces IL-6 protein secretion and gene expression in HFCs through the activation of p38 or p44/42 MAP kinases. These results suggest a potential association between IL-6–mediated inflammatory response and tissue hypertrophy in LSS, and we provide insights into molecular targets for therapeutic interventions targeting LSS-related inflammation through our analysis of the MAP kinase pathway using HFCs