Synergistic gene editing in human iPS cells via cell cycle and DNA repair modulation

Precise gene editing aims at generating single-nucleotide modifications to correct or model human disease. However, precision editing with nucleases such as CRIPSR-Cas9 has seen limited success due to poor efficiency and limited practicality. Here, we establish a fluorescent DNA repair assay in human induced pluripotent stem (iPS) cells to visualize and quantify the frequency of DNA repair outcomes during monoallelic and biallelic targeting. We found that modulating both DNA repair and cell cycle phase via defined culture conditions and small molecules synergistically enhanced the frequency of homology-directed repair (HDR). Notably, targeting in homozygous reporter cells results in high levels of editing with a vast majority of biallelic HDR outcomes. We then leverage efficient biallelic HDR with mixed ssODN repair templates to generate heterozygous mutations. Synergistic gene editing represents an effective strategy to generate precise genetic modifications in human iPS cells

A Genome-Wide Association Study of the Protein C Anticoagulant Pathway

The Protein C anticoagulant pathway regulates blood coagulation by preventing the inadequate formation of thrombi. It has two main plasma components: protein C and protein S. Individuals with protein C or protein S deficiency present a dramatically increased incidence of thromboembolic disorders. Here, we present the results of a genome-wide association study (GWAS) for protein C and protein S plasma levels in a set of extended pedigrees from the Genetic Analysis of Idiopathic Thrombophilia (GAIT) Project. A total number of 397 individuals from 21 families were typed for 307,984 SNPs using the Infinium® 317 k Beadchip (Illumina). Protein C and protein S (free, functional and total) plasma levels were determined with biochemical assays for all participants. Association with phenotypes was investigated through variance component analysis. After correcting for multiple testing, two SNPs for protein C plasma levels (rs867186 and rs8119351) and another two for free protein S plasma levels (rs1413885 and rs1570868) remained significant on a genome-wide level, located in and around the PROCR and the DNAJC6 genomic regions respectively. No SNPs were significantly associated with functional or total protein S plasma levels, although rs1413885 from DNAJC6 showed suggestive association with the functional protein S phenotype, possibly indicating that this locus plays an important role in protein S metabolism. Our results provide evidence that PROCR and DNAJC6 might play a role in protein C and free protein S plasma levels in the population studied, warranting further investigation on the role of these loci in the etiology of venous thromboembolism and other thrombotic diseases

Non-invasive muscle contraction assay to study rodent models of sarcopenia

<p>Abstract</p> <p>Background</p> <p>Age-related sarcopenia is a disease state of loss of muscle mass and strength that affects physical function and mobility leading to falls, fractures, and disability. The need for therapies to treat age-related sarcopenia has attracted intensive preclinical research. To facilitate the discovery of these therapies, we have developed a non-invasive rat muscle functional assay system to efficiently measure muscle force and evaluate the efficacy of drug candidates.</p> <p>Methods</p> <p>The lower leg muscles of anesthetized rats are artificially stimulated with surface electrodes on the knee holders and the heel support, causing the lower leg muscles to push isometric pedals that are attached to force transducers. We developed a stimulation protocol to perform a fatigability test that reveals functional muscle parameters like maximal force, the rate of fatigue, fatigue-resistant force, as well as a fatigable muscle force index. The system is evaluated in a rat aging model and a rat glucocorticoid-induced muscle loss model</p> <p>Results</p> <p>The aged rats were generally weaker than adult rats and showed a greater reduction in their fatigable force when compared to their fatigue-resistant force. Glucocorticoid treated rats mostly lost fatigable force and fatigued at a higher rate, indicating reduced force from glycolytic fibers with reduced energy reserves.</p> <p>Conclusions</p> <p>The involuntary contraction assay is a reliable system to assess muscle function in rodents and can be applied in preclinical research, including age-related sarcopenia and other myopathy.</p

ACE2 knockout hinders SARS-CoV-2 propagation in iPS cell-derived airway and alveolar epithelial cells

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, continues to spread around the world with serious cases and deaths. It has also been suggested that different genetic variants in the human genome affect both the susceptibility to infection and severity of disease in COVID-19 patients. Angiotensin-converting enzyme 2 (ACE2) has been identified as a cell surface receptor for SARS-CoV and SARS-CoV-2 entry into cells. The construction of an experimental model system using human iPS cells would enable further studies of the association between viral characteristics and genetic variants. Airway and alveolar epithelial cells are cell types of the lung that express high levels of ACE2 and are suitable for in vitro infection experiments. Here, we show that human iPS cell-derived airway and alveolar epithelial cells are highly susceptible to viral infection of SARS-CoV-2. Using gene knockout with CRISPR-Cas9 in human iPS cells we demonstrate that ACE2 plays an essential role in the airway and alveolar epithelial cell entry of SARS-CoV-2 in vitro. Replication of SARS-CoV-2 was strongly suppressed in ACE2 knockout (KO) lung cells. Our model system based on human iPS cell-derived lung cells may be applied to understand the molecular biology regulating viral respiratory infection leading to potential therapeutic developments for COVID-19 and the prevention of future pandemics

細胞周期およびDNA修復調節を介したヒトiPS細胞における相乗的遺伝子編集

京都大学0048新制・課程博士博士(医科学)甲第22700号医科博第115号新制||医科||8(附属図書館)京都大学大学院医学研究科医科学専攻(主査)教授 遊佐 宏介, 教授 近藤 玄, 教授 齊藤 博英学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDFA

Study of the benefits of the ToF-SIMS in solving industrial problems on glass pollution: two case studies

Profitability, productivity, reactivity, all these terms have become prime objectives for companies across industries. Manufacturing firms are more than ever subject to pressure in order to maximize production and therefore minimize as much as possible production line failures or defects. AGC Glass Europe is a world leader in production of glass, from windows, to wind- screens, to mirrors, etc. The AGC Technovation Centre, known as the research and de- velopment centre for the firm is located in Gosselies, Charleroi, making from Belgium an important hub for centralizing and solving all production issues faced by AGC Glass Europe. This paper, will look at two recent issues faced by AGC Glass Europe with regards to organic pollution in their production cycle of mirrors as well as windscreens and backlites, in order to assist AGC in preventing these issues to happen in the future. This research will in fact aim at setting a starting point for AGC towards a better understanding of the pollution issues they have encountered, with the objective to offer more information and perspectives for further analysis. Both cases required to recreate the pollution by ”in-lab” manipulations. The ”in-lab” polluted samples were then studied using the very sensitive SIMS surface technique based on time of flight (ToF-SIMS). Indeed, the aim of the study is to determine the contribution of this technique on the pollution detection. Other techniques were used in addition to the SIMS technique, namely infra-red spectrometry and surface tension measurement. ToF-SIMS is a very sensitive tool, and has shown great effectiveness in the two case studies by revealing the compounds responsible for the glass pollution in both cases, namely terpenoids for the first case and additives for the second case study. The in-depth analysis of the second case has also unveiled a ranking in the PE foils according to their polluting capacity. This has been done thanks to the IR analysis. These two cases have been identified as critical by AGC Glass Europe, and have been submitted for review to the AGC Technovation Centre. They will therefore be the main focus of this thesisMaster [120] : ingénieur civil en chimie et science des matériaux, Université catholique de Louvain, 201