CRISPR/Cas9-mediated homology-directed repair (HDR) is used for error-free targeted knock-in of foreign donor DNA. However, the low efficiency of HDR-mediated knock-in hinders establishment of ...knock-in clones. Double-strand breaks (DSBs) induced by CRISPR/Cas9 are preferentially repaired by non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) before HDR can occur, thereby preventing HDR-mediated knock-in. NHEJ/MMEJ also cause random integrations, which give rise to false-positive knock-in events, or silently disrupt the genome. In this study, we optimized an HDR-mediated knock-in method for mouse embryonic stem cells (mESCs). We succeeded in improving efficiency of HDR-mediated knock-in of a plasmid donor while almost completely suppressing NHEJ/MMEJ-based integration by combining in vivo-linearization of the donor plasmid, transient knockdown of DNA polymerase θ, and chemical inhibition of DNA-dependent protein kinase (DNA-PK) by M3814. This method also dramatically improved the efficiency of biallelic knock-in; at the Rosa26a locus, 95% of HDR-mediated knock-in clones were biallelic. We designate this method BiPoD (Biallelic knock-in assisted by Pol θ and DNA-PK inhibition). BiPoD achieved simultaneous efficient biallelic knock-in into two loci. BiPoD, therefore, enables rapid and easy establishment of biallelic knock-in mESC lines.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Although the recently marketed stent retriever thrombectomy devices have demonstrated a high recanalization rate and favorable clinical outcomes, there is a concern about the risks of intimal ...injuries when pulling out the stent in the unfolded position. In this study, the Solitaire Flow Restoration System and the Trevo retriever were used in a histopathological comparison of vascular injuries caused by stent retriever thrombectomy devices.
Rabbit carotid arteries were used in the experiments with stent retriever thrombectomy devices. Carotid artery samples were harvested either 1 or 2 weeks postoperatively for histological examination.
Histological changes caused by the use of stent retriever thrombectomy devices were observed from the intimal to medial layers. With the Solitaire FR 4 mm, intimal and medial thickening was observed 1 week postoperatively, and progression of intimal thickening was observed 2 weeks postoperatively. The extent of intimal thickening tended to be greater with the Solitaire FR 6 mm than with the Solitaire FR 4 mm, but this difference was not significant. Compared with the Solitaire FR 4 mm, the Trevo had a significantly smaller area of intimal thickening.
Although there are some differences among devices, results from this study indicate that stent retriever thrombectomy devices induce vascular damage that extends to the medial layer.
Six new sesquiterpenes, tsukiyols A–C, neoilludin C, and 4-O-methylneoilludins A and B, were isolated from the fruiting body of Omphalotus japonicus (Kawam.) Kirchm. & O. K. Mill. Additionally, six ...known compounds, illudin S, neoilludins A–B, 5-hydroxydichomitol, ergosterolperoxide, and 3β,5α,9α-trihydroxyergosta-7,22-diene-6-one, were also obtained. Their chemical structures were determined with MS, IR, and NMR spectra and the absolute configurations of neoilludins A–C, 4-O-methylneoilludins A, and B were determined with electronic circular dichroism (ECD). Illudin S and 3β,5α,9α-trihydroxyergosta-7,22-diene-6-one showed cytotoxicity against human acute promyelocytic leukemia HL60 cells. Illudin S, 4-O-methylneoilludin A, B, and tsukiyol C showed growth-restoring activity against mutant yeast via Ca2+-signal transduction.
Osimertinib is the standard of care in the initial treatment for advanced epidermal growth factor receptor (EGFR) mutation–positive lung cancer. However, clinical data and reliable prognostic ...biomarkers are insufficient.
We performed a retrospective multicentre cohort study for 538 EGFR mutation–positive patients, who received osimertinib as the initial treatment between August 2018 and December 2019. The main outcome was progression-free survival (PFS).
The median observation period was 14.7 months (interquartile range 11.4–20.0). The median PFS was 20.5 months (95% confidence interval CI 18.6−not reached). Multivariate analysis showed that sex (male) (hazard ratio HR 1.99, 95% CI 1.35–2.93, P = 0.001), malignant effusions (HR 1.51, 95% CI 1.11–2.04, P = 0.008), liver metastasis (HR 1.55, 95% CI 1.03–2.33, P = 0.037), advanced unresectable cases (HR 1.71, 95% CI, 1.04–2.82, P = 0.036), mutation type and programmed cell death-ligand 1 (PD-L1) expression were associated with PFS. The L858R (HR 1.55, 95% CI 1.01–2.38, P = 0.043) and uncommon mutations (HR 3.15, 95% CI 1.70–5.83, P < 0.001) were associated with PFS. PD-L1 expression of 1–49% (HR 1.66, 95% CI 1.05–2.63, P = 0.029), ≥50% (HR 2.24, 95% CI 1.17–4.30, P = 0.015) and unknown (HR 1.53, 95% CI 1.05–2.22, P = 0.026) was associated with PFS. The main reasons for treatment discontinuation among 219 patients were disease progression (44.3%), pneumonitis (25.5%) and other adverse events (16.0%).
During initial treatment with osimertinib, PD-L1 expression is significantly related to PFS. Adverse events are a noteworthy reason for discontinuation.
•Osimertinib is standard treatment for advanced EGFR mutation-positive lung cancer.•Clinical data and reliable prognostic biomarkers remain insufficient.•Tumour PD-L1 expression level is associated with progression-free survival.•Adverse events are a common reason for treatment discontinuation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Patients with non-small cell lung cancer (NSCLC) that harbors epidermal growth factor receptor (EGFR) mutations initially respond to EGFR-tyrosine kinase inhibitors (TKI) but eventually experience ...relapse. Acquired resistance to EGFR-TKIs is strongly associated with patient mortality. Thus, elucidation of the mechanism of acquired resistance to EGFR-TKIs is of great importance. In this study, gefitinib-resistant cell line models were established by long-term exposure to gefitinib using the gefitinib-sensitive lung cancer cell lines, PC9 and HCC827. Expression analyses indicated that both FGFR1 and FGF2 were increased in PC9 gefitinib-resistant (PC9 GR) cells as compared with PC9 naïve (PC9 na) cells. Importantly, proliferation of gefitinib-resistant cells was dependent on the FGF2 -FGFR1 pathway. Mechanistically, inhibition of either FGF2 or FGFR1 by siRNA or FGFR inhibitor (PD173074) restored gefitinib sensitivity in PC9 GR cells. These data suggest that FGF2 -FGFR1 activation through an autocrine loop is a novel mechanism of acquired resistance to EGFR-TKIs.
Computer Generated Holograms (CGH) are generated on computers; however, a great deal of computational power is required because the quality of the image is proportional to the number of point light ...sources of a 3D object. The Wavefront Recording Plane (WRP) method is an algorithm that enables reduction of the amount of calculations required. However, the WRP method also has a defect; it is not effective in the case of a 3D object with a deep structure. In this study, we propose two improved WRP methods: "Least Square Tilted WRP method" and "RANSAC Multi-Tilted WRP method."
OBJECTIVE The durability of embolization of large aneurysms is enhanced by use of the neck-bridging stent. However, it remains unclear what factors contribute to decreased recanalization. The purpose ...of this study was to demonstrate the contribution of the straightening effect of the parent artery to the durability of stent-assisted coiling for large aneurysms. METHODS Of the 182 aneurysms treated by embolization since the introduction of the neurovascular stent, 82 consecutive unruptured aneurysms with a diameter greater than 7 mm were selected. There were 52 aneurysms treated with a stent (Group S) and 30 treated without a stent (Group NS). Occlusion status was evaluated 12 months after embolization with digital subtraction angiography. The vascular angle of the parent artery was measured before, immediately after, and 12 months after embolization. The rates of recanalization were compared between Group S and Group NS. In Group S, the rates of recanalization were further compared between those aneurysms with and without a significant angle change. RESULTS The rate of major recanalization was 9.6% in Group S and 26.7% in Group NS. The volume embolization ratio was 32.6% in Group S and 31.6% in Group NS, with no statistically significant difference. However, the angulation change before and after coiling was significantly higher in Group S (10.6°) than in Group NS (0.9°). The difference in the angulation was more evident 12 months after coiling (19.1° in Group S and 1.5° in Group NS). In Group S, recanalization was found in 14.3% of 35 stented aneurysms without a significant angular change when a significant angular change was defined as more than 20°. In contrast, all 17 aneurysms with ≥ 20° of angular change remained occluded. CONCLUSIONS Significant angular change of ≥ 20° most likely leads to decreased recanalization following stent-assisted embolization of large aneurysms.
EGFR mutated lung cancer accounts for a significant subgroup of non-small-cell lung cancer (NSCLC). Over the last decade, multiple EGFR tyrosine kinase inhibitors (EGFR-TKIs) have been developed to ...target mutated EGFR. However, there is little information regarding mutation specific potency of EGFR-TKIs against various types of EGFR mutations. The purpose of this study is to establish an in vitro model to determine the "therapeutic window" of EGFR-TKIs against various types of EGFR mutations, including EGFR exon 20 insertion mutations. The potency of 1st (erlotinib), 2nd (afatinib) and 3rd (osimertinib and rociletinib) generation EGFR-TKIs was compared in vitro for human lung cancer cell lines and Ba/F3 cells, which exogenously express mutated or wild type EGFR. An in vitro model of mutation specificity was created by calculating the ratio of IC50 values between mutated and wild type EGFR. The in vitro model identified a wide therapeutic window of afatinib for exon 19 deletions and L858R and of osimertinib and rociletinib for T790M positive mutations. The results obtained with our models matched well with previously reported preclinical and clinical data. Interestingly, for EGFR exon 20 insertion mutations, most of which are known to be resistant to 1st and 2nd generation EGFR-TKIS, osimertinib was potent and presented a wide therapeutic window. To our knowledge, this is the first report that has identified the therapeutic window of osimertinib for EGFR exon 20 insertion mutations. In conclusion, this model will provide a preclinical rationale for proper selection of EGFR-TKIs against clinically-relevant EGFR mutations.
Fast hologram calculation methods are critical in real-time holography applications such as three-dimensional (3D) displays. We recently proposed a wavelet transform-based hologram calculation called ...WASABI. Even though WASABI can decrease the calculation time of a hologram from a point cloud, it increases the calculation time with increasing propagation distance. We also proposed a wavefront recoding plane (WRP) method. This is a two-step fast hologram calculation in which the first step calculates the superposition of light waves emitted from a point cloud in a virtual plane, and the second step performs a diffraction calculation from the virtual plane to the hologram plane. A drawback of the WRP method is in the first step when the point cloud has a large number of object points and/or a long distribution in the depth direction. In this paper, we propose a method combining WASABI and the WRP method in which the drawbacks of each can be complementarily solved. Using a consumer CPU, the proposed method succeeded in performing a hologram calculation with 2048×2048 pixels from a 3D object with one million points in approximately 0.4s.
•Fast hologram calculation methods are critical in real-time holography applications.•We propose a method combining WASABI and the WRP method.•A hologram from a 3D object with one million points is calculated in 0.4s.
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