Capture of linear fragments at a double-strand break in yeast

Double-strand breaks (DSBs) are dangerous chromosomal lesions that must be efficiently repaired in order to avoid loss of genetic information or cell death. In all organisms studied to date, two different mechanisms are used to repair DSBs: homologous recombination (HR) and non-homologous end joining (NHEJ). Previous studies have shown that during DSB repair, non-homologous exogenous DNA (also termed ‘filler DNA’) can be incorporated at the site of a DSB. We have created a genetic system in the yeast Saccharomyces cerevisiae to study the mechanism of fragment capture. Our yeast strains carry recognition sites for the HO endonuclease at a unique chromosomal site, and plasmids in which a LEU2 gene is flanked by HO cut sites. Upon induction of the HO endonuclease, a linear extrachromosomal fragment is generated in each cell and its incorporation at the chromosomal DSB site can be genetically monitored. Our results show that linear fragments are captured at the repaired DSB site at frequencies of 10−6 to 10−4 per plated cell depending on strain background and specific end sequences. The mechanism of fragment capture depends on the NHEJ machinery, but only partially on the homologous recombination proteins. More than one fragment can be used during repair, by a mechanism that relies on the annealing of small complementary sequences. We present a model to explain the basis for fragment capture

The study of an effect on the motor learning of equilibrium functions in pre-school children

We made a new apparatus, named the Balance meter, to make it clear that pre-school children show the states of equilibrium functions, which are remarkable for development of a motor nerve, the properties of this apparatus possess both the role of a measurement system and of a playmaterials. By using this, therefore, it is interested in that how balance will be performed. To investigate the problems, two similar groups of 5 subjects at four and five years of age were formed, (especialy within ± 1/2 σ in body height, weight and behaviaral attitudes, etc.) Moreover the some acquired states of equilibrium functions were measured after a week the motor learning task was over. The results obtained in this study are as follows; 1) The reliability of a new upparatus is recognized from the results which showed asignificant correlation in the case of the first time as well as the second measuremant. (r=.802) 2) The experimental groups showed rapidly improvement at the records of the Balance meter, while, the control groups were not found out some changes at the behaviaral attitudes ; we say that equilibrium functons in pre-school children is educability, trainability, and it isn\u27t unchangeability. 3) The performance process of the motor learning at five years of age is greater the effect than that at four, and females than males. From those results, we.say that in this time children have to be dealed with the methods of the level of their development

Proximal (entry) tear of dissecting aortic aneurysm visualized by three-dimensional echocardiography

AbstractJ Thorac Cardiovasc Surg 2002;124:1245-

In vitro methods to ensure absence of residual undifferentiated human induced pluripotent stem cells intermingled in induced nephron progenitor cells

ヒトiPS細胞から作製した腎前駆細胞に未分化な細胞が残存していないことを確認する方法の開発. 京都大学プレスリリース. 2022-11-16.A new sensitive method to detect for minute amounts of contaminating undifferentiated iPS cells. 京都大学プレスリリース. 2022-11-21.Cell therapies using human induced pluripotent stem cell (hiPSC)-derived nephron progenitor cells (NPCs) are expected to ameliorate acute kidney injury (AKI). However, using hiPSC-derived NPCs clinically is a challenge because hiPSCs themselves are tumorigenic. LIN28A, ESRG, CNMD and SFRP2 transcripts have been used as a marker of residual hiPSCs for a variety of cell types undergoing clinical trials. In this study, by reanalyzing public databases, we found a baseline expression of LIN28A, ESRG, CNMD and SFRP2 in hiPSC-derived NPCs and several other cell types, suggesting LIN28A, ESRG, CNMD and SFRP2 are not always reliable markers for iPSC detection. As an alternative, we discovered a lncRNA marker gene, MIR302CHG, among many known and unknown iPSC markers, as highly differentially expressed between hiPSCs and NPCs, by RNA sequencing and quantitative RT-PCR (qRT-PCR) analyses. Using MIR302CHG as an hiPSC marker, we constructed two assay methods, a combination of magnetic bead-based enrichment and qRT-PCR and digital droplet PCR alone, to detect a small number of residual hiPSCs in NPC populations. The use of these in vitro assays could contribute to patient safety in treatments using hiPSC-derived cells

Chromatin profiling identifies chondrocyte-specific Sox9 enhancers important for skeletal development

Ichiyama-Kobayashi S., Hata K., Wakamori K., et al. Chromatin profiling identifies chondrocyte-specific Sox9 enhancers important for skeletal development. JCI Insight 9, e175486 (2024); https://doi.org/10.1172/jci.insight.175486.The transcription factor SRY-related HMG box 9 (Sox9) is essential for chondrogenesis. Mutations in and around SOX9 cause campomelic dysplasia (CD) characterized by skeletal malformations. Although the function of Sox9 in this context is well studied, the mechanisms that regulate Sox9 expression in chondrocytes remain to be elucidated. Here, we have used genome-wide profiling to identify 2 Sox9 enhancers located in a proximal breakpoint cluster responsible for CD. Enhancer activity of E308 (located 308 kb 5′ upstream) and E160 (located 160 kb 5′ upstream) correlated with Sox9 expression levels, and both enhancers showed a synergistic effect in vitro. While single deletions in mice had no apparent effect, simultaneous deletion of both E308 and E160 caused a dwarf phenotype, concomitant with a reduction of Sox9 expression in chondrocytes. Moreover, bone morphogenetic protein 2–dependent chondrocyte differentiation of limb bud mesenchymal cells was severely attenuated in E308/E160 deletion mice. Finally, we found that an open chromatin region upstream of the Sox9 gene was reorganized in the E308/E160 deletion mice to partially compensate for the loss of E308 and E160. In conclusion, our findings reveal a mechanism of Sox9 gene regulation in chondrocytes that might aid in our understanding of the pathophysiology of skeletal disorders

Detection of EGFR Gene Mutations Using the Wash Fluid of CT-Guided Biopsy Needle in NSCLC Patients

IntroductionIn this study, we examined whether epidermal growth factor receptor (EGFR) mutations were detectable using a polymerase chain reaction-based assay and wash fluid of computed tomography (CT)-guided lung biopsy needles.MethodsDNA was extracted from wash fluid of CT-guided biopsy needles of 53 lung tumors (as diagnosed according to the results of the CT-guided biopsies). EGFR mutations, specifically exon19 deletions and exon21 L858R mutations, were examined using a mutant-enriched polymerase chain reaction assay. We also examined the presence of EGFR mutations in 26 surgically resected tumor specimens and compared the results with those obtained for the corresponding wash fluid samples.ResultsThe amount of DNA obtained for the wash fluid of the CT-guided biopsy needles ranged from 35 to 2360 ng. There were no significant differences in the amount of extracted DNA according to the tumor characteristics, including tumor size and the percentage of ground glass opacity. Thirty-four of the 53 lung tumor samples were histologically diagnosed as non-small cell lung cancer (NSCLC). Exon19 deletions and exon21 L858R mutations in EGFR were detected in 4 (12%) and 13 (38%) of 34 NSCLC cases, respectively. No EGFR mutations were found in the non-NSCLC cases. The EGFR mutation status in the wash fluid samples was consistent with those obtained for all 26 corresponding surgical specimens.ConclusionOur results indicate that EGFR mutations can be detected using wash fluid of CT-guided biopsy needles. In this manner, the DNA genotype can be determined even in extremely small clinical specimens using highly sensitive assays

Cooling Stability Test of MgB₂ Wire Immersed in Liquid Hydrogen under External Magnetic Field

11th European Conference on Applied Superconductivity (EUCAS2013)Liquid hydrogen (LH₂), which has large latent heat, low viscosity coefficient, is expected to be a candidate for a cryogen for superconducting wires, not only MgB₂ but also other HTC superconductors. LH₂ cooled superconducting wires are expected to have excellent electro-magnetic characteristics, which is necessary to be clear for cooling stability design of LH₂ cooled superconducting device, however, due to handling difficulties of LH₂, there are only few papers on the properties of LH₂ cooled superconductors, especially under external magnetic field. We designed and made an experimental setup which can energize superconducting wires immersed in LH₂ with the current of up to 500A under the condition of external magnetic field up to 7 T and pressure up to 1.5 MPa. In order to confirm experimental method and safety operation of the setup, over current tests were carried out using MgB₂ superconducting wires under various external magnetic field conditions. Critical current of the test wire at the temperature 21, 24, 27, 29 K under external magnetic fields up to 1.2 T was successfully measured. The resistance of the wire also was measured, while the transport current exceeded the critical current of the wire

Non-BAC Component but not Epidermal Growth Factor Receptor Gene Mutation is Associated with Poor Outcomes in Small Adenocarcinoma of the Lung

ObjectiveThe purpose of this study was to identify risk factors for poor clinical outcome after surgical resection of small lung adenocarcinoma.Materials and MethodsClinical records of 127 patients who had pathologic stage IA lung adenocarcinoma 20 mm or less and who had undergone a lobectomy with mediastinal lymph node dissection were reviewed. The percentage of non-bronchioloalveolar carcinoma (non-BAC) components quantified objectively, and epidermal growth factor receptor gene (EGFR) mutation determined by polymerase chain reaction-based assay were retrospectively linked with clinical data.ResultsBased on the percentage of non-BAC component, 127 patients were classified as follows: 26 in group I, BAC, 46 in group II mixed subtype with ≥ 50% BAC, 18 in group III, mixed subtype with under 50% BAC, and 37 in group IV, mixed subtype with all non-BAC components or a pure pattern of one of the non-BAC components. Groups I and II were considered to be a “low non-BAC component type” and groups III and IV were considered to be a “high non-BAC component type.” EGFR mutations in exon19 and exon21 were observed in 64 patients (50.4%). In terms of recurrence, the high non-BAC component type was the only independent factor for recurrence (p = 0.029). Regarding survival, the high age (p = 0.028) and high non-BAC component type (p = 0.046) were independent risk factors for poor overall survival. They were also independent risk factors for poor disease-free survival (p = 0.025 and p = 0.027, respectively).ConclusionThe high non-BAC component but not EGFR mutation status, is an independent risk factor for both recurrence and poor prognosis in patients with stage IA lung adenocarcinoma ≤20 mm

Difference between carbohydrate antigen 19-9 and fluorine-18 fluorodeoxyglucose positron emission tomography in evaluating the treatment efficacy of neoadjuvant treatment in patients with resectable and borderline resectable pancreatic ductal adenocarcinoma: Results of a dual-center study

kita, H, Takahashi, H, Eguchi, H, et al. Difference between carbohydrate antigen 19‐9 and fluorine‐18 fluorodeoxyglucose positron emission tomography in evaluating the treatment efficacy of neoadjuvant treatment in patients with resectable and borderline resectable pancreatic ductal adenocarcinoma: Results of a dual‐center study. Ann Gastroenterol Surg. 2020; 00: 1– 9. https://doi.org/10.1002/ags3.12418