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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 ( Bi allelic knock-in assisted by Po l θ and D NA-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.

Most multigene mutation tests require tissue specimens. However, cytological specimens are easily obtained in the clinical practice and provide high‐quality DNA and RNA. We aimed to establish a test that utilizes cytological specimens and performed a multi‐institutional study to investigate the performance of MINtS, a test based on next‐generation sequencing. A standard procedure for specimen isolation was defined. The specimens were considered suitable for the test if >100 ng DNA and >50 ng RNA could be extracted from them. In total, 500 specimens from 19 institutions were investigated. MINtS detected druggable mutations in 63% (136 of 222) of adenocarcinomas. Discordant results between MINtS and the companion diagnostics were observed in 14 of 310 specimens for the EGFR gene, and 6 of 339 specimens for the ALK fusion genes. Confirmation by other companion diagnostics for the EGFR mutations or the clinical response to an ALK inhibitor all supported the results obtained by MINtS. MINtS along with the isolation procedure presented in the current study will be a platform to establish multigene mutation tests that utilize cytological specimens. UMIN000040415. The performance of a multigene test MINtS that uses cytological specimens was tested as a multi‐institutional study executed as Advanced Medicine A, Japan. MINtS displayed an excellent performance and will be a platform to establish multigene mutation tests that utilize cytological specimens.

Background and objectivesAlthough 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.MethodsRabbit 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.ResultsHistological 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.ConclusionsAlthough 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.

Ramucirumab plus docetaxel (RD) can cause febrile neutropenia (FN), which frequently requires the prophylactic administration of pegfilgrastim. However, the effects of prophylactic pegfilgrastim on FN prevention, therapeutic efficacy, and prognosis after RD have not been fully evaluated in patients with advanced non-small-cell lung cancer (NSCLC). Two hundred and eighty-eight patients with advanced NSCLC who received RD as second-line therapy after platinum-based chemotherapy plus PD-1 blockade were included. Patients were divided into groups with and without prophylactic pegfilgrastim, and adverse events, efficacy, and prognosis were compared between both groups. Of the 288 patients, 247 received prophylactic pegfilgrastim and 41 did not. The frequency of grade 3/4 neutropenia was 62 patients (25.1%) in the pegfilgrastim group and 28 (68.3%) in the control group ( p  < 0.001). The frequency of FN was 25 patients (10.1%) in the pegfilgrastim group and 10 (24.4%) in the control group ( p  = 0.018). The objective response rate was 31.2% and 14.6% in the pegfilgrastim and control groups ( p  = 0.039), respectively. The disease control rate was 72.9% in the pegfilgrastim group and 51.2% in the control group ( p  = 0.009). Median progression free survival was 4.3 months in the pegfilgrastim group and 2.5 months in the control group ( p  = 0.002). The median overall survival was 12.8 and 8.1 months in the pegfilgrastim and control groups ( p  = 0.004), respectively. Prophylactic pegfilgrastim for RD reduced the frequency of grade 3/4 neutropenia and febrile neutropenia and did not appear to be detrimental to patient outcome RD. Clinical Trial Registration Number: UMIN000042333.

We report here newly discovered O -linked-N-acetylglucosamine ( O -GlcNAc) modification of histone H2A at Ser 40 (H2AS40Gc). The mouse genome contains 18 H2A isoforms, of which 13 have Ser 40 and the other five have Ala 40 . The combination of production of monoclonal antibody and mass spectrometric analyses with reverse-phase (RP)-high performance liquid chromatography (HPLC) fractionation indicated that the O -GlcNAcylation is specific to the Ser 40 isoforms. The H2AS40Gc site is in the L1 loop structure where two H2A molecules interact in the nucleosome. Targets of H2AS40Gc are distributed genome-wide and are dramatically changed during the process of differentiation in mouse trophoblast stem cells. In addition to the mouse, H2AS40Gc was also detected in humans, macaques and cows, whereas non-mammalian species possessing only the Ala 40 isoforms, such as silkworms, zebrafish and Xenopus showed no signal. Genome database surveys revealed that Ser 40 isoforms of H2A emerged in Marsupialia and persisted thereafter in mammals. We propose that the emergence of H2A Ser 40 and its O-GlcNAcylation linked a genetic event to genome-wide epigenetic events that correlate with the evolution of placental animals.

ABT‐263 (Navitoclax) is a BH3‐mimetic drugs targeting anti‐apoptotic B‐cell lymphoma‐2 (BCL‐2) family proteins, including BCL‐2, BCL‐xL, and BCL‐w, thereby inducing apoptosis. In small‐cell lung cancer (SCLC) cells, the response to ABT‐263 is associated with the expression of myeloid cell leukemia‐1 (MCL‐1) protein, however the efficacy of ABT‐263 in non‐small‐cell lung cancer (NSCLC) has not been thoroughly evaluated. There are currently no established biomarkers for predicting the efficacy of ABT‐263 treatment in NSCLC. We screened a panel of different NSCLC cell lines and found that ABT‐263 inhibited cell proliferation and induced apoptosis in Calu‐1, Calu‐3, and BID007 cells. Inconsistent with previous reports on SCLC, low levels of MCL‐1 did not predict the response to ABT‐263 in NSCLC cells, however we found that intracellular levels of reactive oxygen species (ROS) in cancer cells were associated with sensitivity to ABT‐263 in NSCLC cells. We also showed that increasing the level of intracellular ROS could enhance the sensitivity to ABT‐263 in NSCLC cells. In summary, we propose that the intracellular levels of ROS could be used as a potential novel biomarker for predicting a response to ABT‐263 in NSCLC. Furthermore, we show some evidence supporting the further assessment of ABT‐263 as a new therapeutic strategy in patients with NSCLC combined with agents regulating ROS levels. We believe that our findings and follow‐up studies on this matter would lead to novel diagnostic and treatment strategies in patients with NSCLC. This article clarified the potential relationship between intracellular ROS and sensitivity to BH3‐mimetic drug, ABT‐263 in non‐small‐cell lung cancer.

Mutations in the gap junction β2 (GJB2) gene, which encodes connexin 26, are the leading cause of genetic deafness. These mutations are characterized by the degeneration and fragmentation of gap junctions and gap junction plaques (GJPs) composed of connexin 26. Dominant-negative mutations of GJB2, such as R75W, cause syndromic hearing loss and palmoplantar keratoderma. We previously reported that the R75W mutation, a single-base substitution where C is replaced by T, causes fragmentation of GJPs. Therefore, an adenine base editor (ABE), which enables A-to-G base conversions, can potentially be useful for the treatment of this genetic disease. Here, we report that an all-in-one adeno-associated virus (AAV) vector, which includes a compact ABE (SaCas9-NNG-ABE8e) with broad targeting range, and a sgRNA targeting the R75W mutation in GJB2 corrected this pathogenic mutation and facilitated the recovery of the gap junction intercellular communication network of GJPs. In a transgenic mouse model with the GJB2 R75W mutation, AAV-mediated base editing also restored the fragmented GJPs to orderly outlines in cochlear supporting cells. Our findings suggest that an ABE-based base-editing strategy could be an optimal treatment for the dominant form of GJB2-related hearing loss, GJB2-related skin diseases, and other deafness-related mutations, especially single-base substitutions.

Genome-wide RNA interference (RNAi) with pooled and barcoded short-hairpin RNA (shRNA) libraries provides a powerful tool for identifying cellular components that are relevant to the modes/mechanisms of action (MoA) of bioactive compounds. shRNAs that affect cellular sensitivity to a given compound can be identified by deep sequencing of shRNA-specific barcodes. We used multiplex barcode sequencing technology by adding sample-specific index tags to PCR primers during sequence library preparation, enabling parallel analysis of multiple samples. An shRNA library screen with this system revealed that downregulation of ATP1A1, an α-subunit of Na + /K + ATPase, conferred significant sensitivity to aurilide B, a natural marine product that induces mitochondria-mediated apoptosis. Combined treatment with ouabain which inhibits Na + /K + ATPase by targeting α-subunits potentiated sensitivity to aurilide B, suggesting that ATP1A1 regulates mitochondria-mediated apoptosis. Our results indicate that multiplex sequencing facilitates the use of pooled shRNA library screening for the identification of combination drug therapy targets.

We identified transmembrane protease, serine 4 (TMPRSS4) as a putative, druggable target by screening surgically resected samples from 90 Japanese non‐small‐cell lung cancer (NSCLC) patients using cDNA microarray. TMPRSS4 has two druggable domains and was upregulated in 94.5% of the lung cancer specimens. Interestingly, we found that TMPRSS4 expression was associated with tissue factor pathway inhibitor 2 (TFPI‐2) expression in these clinical samples. In contrast to TMPRSS4, TFPI‐2 expression was downregulated in NSCLC samples. The in vitro induction of TFPI‐2 in lung cancer cell lines decreased the expression of TMPRSS4 mRNA levels. Reporter assay showed that TFPI‐2 inhibited transcription of TMPRSS4, although partially. Knockdown of TMPRSS4 reduced the proliferation rate in several lung cancer cell lines. When lung cancer cell lines were treated with 5‐aza‐2′‐deoxycytidine or trichostatin A, their proliferation rate and TMPRSS4 mRNA expression levels were also reduced through the upregulation of TFPI‐2 by decreasing its methylation in vitro. The TFPI‐2 methylation level in the low TMPRSS4 group appeared to be significantly low in NSCLC samples (P = 0.02). We found a novel molecular mechanism that TFPI‐2 negatively regulates cell growth by inhibiting transcription of TMPRSS4. We suggest that TMPRSS4 is upregulated by silencing of TFPI‐2 through aberrant DNA methylation and contributes to oncogenesis in NSCLC. TMPRSS4 was up‐regulated by silencing of TFPI‐2 through aberrant DNA methylation and contributed to oncogenesis in NSCLC. It will provide novel therapeutic potential for NSCLC patients by suppressing TMPRSS4 through inhibiting TMPRSS4 directly or indirectly through demethylating TFPI‐2.

Chronic inflammation is involved in the formation and enlargement of cerebral aneurysms (CAs), with macrophages playing a key role in the process. The present study evaluated visualization of macrophages present in CAs using an activatable fluorescent probe (IONP-ICG) comprising an iron oxide nanoparticles (IONPs) conjugated with indocyanine green (ICG). IONP-ICG was intravenously administered to 15-week-old CA model rats ( = 8), and near-infrared fluorescence (NIRF) imaging and histological assessment of exposed CAs and cerebral arteries were performed 48 h later. Similar evaluations were performed in the control group, which included CA model rats given IONPs or ICG ( = 8 each). ICG-derived NIRF signals were detected in three IONP-ICG group rats but not in IONP or ICG control groups. Among the three rats that exhibited signals, NIRF signal accumulation was observed in the CA of two rats and at the site of hemodynamic stress in the left posterior cerebral artery in one rat. Histologically, NIRF signals correlated strongly with macrophage localization. A total of 13 CAs formed in the IONP-ICG group. The number of macrophages in the CA wall was significantly greater in the two CAs that exhibited NIRF signals compared to the remaining 11 CAs that did not ( = 0.037). Moreover, all 11 CAs that did not exhibit NIRF signals were iron-negative, while the two CAs that exhibited NIRF signals were both iron-positive ( = 0.013). NIRF imaging using an activatable IONP-ICG probe is feasible for detecting the macrophage-rich regions in CAs and the cerebral artery wall, which is considered an early lesion in the process of CA formation.