Highly efficient heritable targeted deletions of gene clusters and non-coding regulatory regions in Arabidopsis using CRISPR/Cas9

Genome editing using CRISPR/Cas9 is considered the best instrument for genome engineering in plants. This methodology is based on the nuclease activity of Cas9 that is guided to specific genome sequences by single guide RNAs (sgRNAs) thus enabling researchers to engineer simple mutations or large chromosomal deletions. Current methodologies for targeted genome editing in plants using CRISPR/Cas9 are however largely inefficient, mostly due to low Cas9 activity, variable sgRNA efficiency and low heritability of genetic lesions. Here, we describe a newly developed strategy to enhance CRISPR/Cas9 efficiency in Arabidopsis thaliana focusing on the design of novel binary vectors (pUbiCAS9-Red and pEciCAS9-Red), the selection of highly efficient sgRNAs, and the use of direct plant regeneration from induced cell cultures. Our work demonstrates that by combining these three independent developments, heritable targeted chromosomal deletions of large gene clusters and intergenic regulatory sequences can be engineered at a high efficiency. Our results demonstrate that this improved CRISPR/Cas9 methodology can provide a fast, efficient and cost-effective tool to engineer targeted heritable chromosomal deletions, which will be instrumental for future high-throughput functional genomics studies in plants

Tenomodulin expression in the periodontal ligament enhances cellular adhesion.

Tenomodulin (Tnmd) is a type II transmembrane protein characteristically expressed in dense connective tissues such as tendons and ligaments. Its expression in the periodontal ligament (PDL) has also been demonstrated, though the timing and function remain unclear. We investigated the expression of Tnmd during murine tooth eruption and explored its biological functions in vitro. Tnmd expression was related to the time of eruption when occlusal force was transferred to the teeth and surrounding tissues. Tnmd overexpression enhanced cell adhesion in NIH3T3 and human PDL cells. In addition, Tnmd-knockout fibroblasts showed decreased cell adhesion. In the extracellular portions of Tnmd, the BRICHOS domain or CS region was found to be responsible for Tnmd-mediated enhancement of cell adhesion. These results suggest that Tnmd acts on the maturation or maintenance of the PDL by positively regulating cell adhesion via its BRICHOS domain

Исследование процесса утилизации сточных вод фабрики по переработке ильменитового концентрата, производительностью 10000 т/год по концентрату

Объектом исследования является процесса утилизации сточных вод фабрики по переработке ильменитового концентрата производительностью 10000 т/год по концентрату. Цель работы – спроектировать участок очистки технической воды. В процессе исследования проводились эксперименты по выпариванию раствора фторид аммония. В результате исследования определены оптимальные параметры введения процесса. Рaссчитaны основные технико-экономические покaзaтели (себестоимость переделa 108294133,24)The object of research is the process of recycling waste water plant for the processing of ilmenite concentrate capacity of 10,000 tons / year for the concentrate. The purpose of the work - design of industrial water purification station. The study carried out experiments on the evaporation of a solution of ammonium fluoride. The study determined the optimal parameters of the process of administration. Rasschitany basic technical and economic pokazateli (peredela 108,294,133.24 cost

組織因子惹起-トロンボモジュリン添加凝固波形解析を用いたプロテインC経路異常を伴う血栓性素因のスクリーニング法

Objectives: Absolute or relative protein (P)C pathway abnormalities (PC deficiency, PS deficiency, antiphospholipid syndrome (APS), factor (F)V-abnormality, and high FVIII level) cause thrombophilia. Although screening assays for these thrombophilias are available, one utilizing clot waveform analysis (CWA) remains unknown. We aimed to establish a CWA-based screening assay to distinguish PC pathway abnormality-related thrombophilia. Methods: Samples were reacted with tissue factor (TF)/phospholipids and recombinant thrombomodulin (rTM; optimal 20 nM), followed by CWA measurement. The peak ratio (with/without rTM) of the first derivative curve of clot waveform was calculated. Results: The peak ratio in healthy plasmas (n = 35) was 0.36 ± 0.13; hence, the cutoff value was set to 0.49. The peak ratios in plasmas with PC deficiency, PS deficiency, high-FVIII (spiked 300 IU/dl), and APS were higher than the cutoff values (0.79/0.97/0.50/0.93, respectively). PC-deficient plasma or PS-deficient plasma mixed with normal plasma (25%/50%/75%/100% PC or PS level) showed dose-dependent decreases in the peak ratios (PC deficient: 0.85/0.64/0.44/0.28; PS deficient: 0.69/0.53/0.40/0.25), suggesting that the peak ratio at ≤50% of PC or PS level exceeded the cutoff value. The peak ratio in FV deficiency with FV ≤25% was higher than the cutoff value. FV-deficient plasma spiked with 40 IU/dl rFV-R506Q (FVLeiden ) or rFV-W1920R (FVNara ) showed >90% peak ratios. Conclusions: rTM-mediated TF-triggered CWA might be useful for screening PC pathway abnormality-related thrombophilia.博士（医学）・乙第1527号・令和4年9月28日© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.This is the peer reviewed version of the following article: [https://onlinelibrary.wiley.com/doi/10.1111/ejh.13777], which has been published in final form at [https://doi.org/10.1111/ejh.13777]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited.発行元が定める登録猶予期間終了の後、本文を登録予定（2023.07

浸潤および血管新生を通しての人肝細胞癌の進行におけるケラチン19分子の役割

BACKGROUND: Keratin (K) 19-positive hepatocellular carcinoma (HCC) is well known to have a higher malignant potential than K19-negative HCC: However, the molecular mechanisms involved in K19-mediated progression of HCC remain unclear. We attempted to clarify whether K19 directly affects cell survival and invasiveness in association with cellular senescence or epithelial-mesenchymal transition (EMT) in K19-positive HCC. METHODS: K19 expression was analysed in 136 HCC surgical specimens. The relationship of K19 with clinicopathological factors and survival was analysed. Further, the effect of K19 on cell proliferation, invasion, and angiogenesis was examined by silencing K19 in the human HCC cell lines, HepG2, HuH-7, and PLC/PRF/5. Finally, we investigated HCC invasion, proliferation, and angiogenesis using K19-positive HCC specimens. RESULTS: Analysis of HCC surgical specimens revealed that K19-positive HCC exhibited higher invasiveness, metastatic potential, and poorer prognosis. In vitro experiments using the human HCC cell lines revealed that K19 silencing suppressed cell growth by inducting apoptosis or upregulating p16 and p27, resulting in cellular senescence. In addition, transfection with K19 siRNA upregulated E-cadherin gene expression, significantly inhibited the invasive capacity of the cells, downregulated angiogenesis-related molecules such as vasohibin-1 (VASH1) and fibroblast growth factor 1 (FGFR1), and upregulated vasohibin-2 (VASH2). K19-positive HCC specimens exhibited a high MIB-1 labelling index, decreased E-cadherin expression, and high microvessel density around cancer foci. CONCLUSION: K19 directly promotes cancer cell survival, invasion, and angiogenesis, resulting in HCC progression and poor clinical outcome. K19 may therefore be a novel drug target for the treatment of K19-positive HCC.博士（医学）・乙第1399号・平成29年3月15日© The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated

CORONA, PHABULOSA AND PHAVOLUTA collaborate with BELL1 to confine WUSCHEL expression to the nucellus in Arabidopsis ovules

Angiosperm ovules consist of three proximal-distal domains – the nucellus, chalaza and funiculus – demarcated by developmental fate and specific gene expression. Mutation in three paralogous class III homeodomain leucine zipper (HD-ZIPIII) genes leads to aberrations in ovule integument development. Expression of WUSCHEL (WUS) is normally confined to the nucellar domain, but in this triple mutant expression expands into the chalaza. MicroRNA-induced suppression of this expansion partially suppresses the effects of the HD-ZIPIII mutations on ovule development, implicating ectopic WUS expression as a component of the mutant phenotype. bell1 (bel1) mutants produce aberrant structures in place of the integuments and WUS is ectopically expressed in these structures. Combination of bel1 with the HD-ZIPIII triple mutant leads to a striking phenotype in which ectopic ovules emerge from nodes of ectopic WUS expression along the funiculi of the primary ovules. The synergistic phenotype indicates that BEL1 and the HD-ZIPIII genes act in at least partial independence in confining WUS expression to the nucellus and maintaining ovule morphology. The branching ovules of the mutant resemble those of some fossil gymnosperms, implicating BEL1 and HD-ZIPIII genes as players in the evolution of the unbranched ovule form in extant angiosperms. © 2016. Published by The Company of Biologists Ltd.Embargo Period 12 month

慢性肺疾患の早産児におけるプロテインC経路 : 前向き研究

Background: Chronic lung disease (CLD) is a major neonatal pulmonary disorder associated with inflammation. Recent studies have shown that protein C anticoagulant pathways, such as those for protein C (PC), protein S (PS), and thrombomodulin (TM), could be useful indices for reflecting pulmonary injury. However, the involvement of these factors in preterm infants with very low birthweight (VLBW) who have developed CLD remains to be investigated. Here, we investigated whether PC pathway-related factors could predict the development of CLD in preterm infants with VLBW. Methods: We collected plasma samples from 26 preterm infants with VLBW (13 each from those with and without CLD) at the time of birth and measured TM, PC, and PS levels in their plasmas. We analyzed prospectively the relationship between these factors in infants with and without CLD. Results: There were significant differences in gestational age, birthweight, Apgar score (5 min), and duration of mechanical ventilation between the CLD and non-CLD groups. No significant differences in the PC and PS levels at birth were observed between the two groups, whereas the TM levels in the CLD group were significantly higher than those in the non-CLD group (P = 0.013). The TM levels correlated with gestational age and duration of mechanical ventilation. However, covariance analysis demonstrated that gestational age was significantly associated with TM levels, and consequently, development of CLD was not associated with TM level at birth. Conclusions: Thrombomodulin, PC, and PS levels at birth could not predict the development of CLD in preterm infants with VLBW.博士（医学）・甲第850号・令和4年9月28日© 2022 Japan Pediatric Society.This is the peer reviewed version of the following article: [https://onlinelibrary.wiley.com/doi/10.1111/ped.15221], which has been published in final form at [https://doi.org/10.1111/ped.15221]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited.発行元が定める登録猶予期間終了の後、本文を登録予定（2023.01

日本における小児血友病患者の日本語版KIDSCREEN-52を用いたQOLの自己評価および保護者による評価

Introduction: Assessing health-related quality of life (HRQOL) is critical for providing comprehensive clinical care to patients with haemophilia. HRQOL in individuals with similar cultural backgrounds should be compared using internationally standardized, generic questionnaires. Aim: To evaluate self-/parent-assessed HRQOL in Japanese children and adolescents with haemophilia A or B. Methods: Children and adolescents aged 8-18 years were enrolled. The haemophilia group comprised families with haemophilia, and the control group comprised those without chronic illness. HRQOL was assessed using the self-/parent-reported questionnaire KIDSCREEN-52, the Japanese version. The Oslo 3-Item Social Support Scale was investigated. Results: The questionnaire was completed by 36 families in the haemophilia group and 160 parents and children in the control group. Haemophilia children aged 8-12 years had lower scores for 'moods and emotions' than control children; the parents had lower scores in the haemophilia group than in the control group for 'moods and emotions', 'social support and peers', and 'school environment'. No significant differences in HRQOL were observed between both groups of adolescents aged 13-18 years or their parents. Neck-shoulder pain was associated with a low psychological state, including 'self-perception', but other joint pains did not affect the outcomes of the HRQOL indices. Social support weaknesses were associated with low physical and psychological states, whereas unexpected hospital visits identified low values for 'self-perception', 'autonomy', and 'school environment'. Conclusion: Proactive mental and clinical care in haemophilia families, especially with young children, will foster a better environment for patients and their parents and ease concerns about progress in haemophilia.博士（医学）・甲第747号・令和2年6月30日© 2020 John Wiley & Sons Ltd.This is the peer reviewed version of the following article: [https://onlinelibrary.wiley.com/doi/full/10.1111/hae.13945], which has been published in final form at [https://doi.org/10.1111/hae.13945]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions

ヒト膵癌におけるNectin-4発現の腫瘍増殖、血管新生に対する効果と臨床的意義

BACKGROUND: Nectin-4 belongs to the nectin family that has diverse physiological and pathological functions in humans. Recent studies have also suggested some roles for Nectin-4 in several human cancers. However, the precise roles and clinical relevance of Nectin-4 in tumors are largely unknown. METHODS: Nectin-4 expression was investigated in 123 patients with pancreatic cancer by immunohistochemistry. Furthermore, we investigated the association of Nectin-4 in pancreatic cancer with tumor proliferation, angiogenesis and immunity by using immunohistochemistry and siRNA interference method. RESULTS: Patients with high Nectin-4 expression had poorer postoperative prognosis than those with low expression. Importantly, multivariate analysis indicated that Nectin-4 expression had a significant independent prognostic value in pancreatic cancer (HR = 1.721, 1.085-2.730; P = 0.021). Tumor Nectin-4 expression was significantly correlated with Ki67 expression. In addition, siRNA-mediated gene silencing of Nectin-4 significantly inhibited the cell proliferation in human pancreatic cancer cells, Capan-2 and BxPC-3. Furthermore, Nectin-4 expression was also positively correlated with VEGF expression and intratumoral microvessel density. However, there were no significant correlations of tumor Nectin-4 expression with tumor-infiltrating T cells. CONCLUSION: Nectin-4 is a significant prognostic predictor, and may play a critical role in pancreatic cancer. Nectin-4 may be novel therapeutic target for pancreatic cancer.博士（医学）・乙第1400号・平成29年6月28日Copyright © Nishiwada et al.; licensee BioMed Central. 2015 : This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated