Generation of hypoimmunogenic induced pluripotent stem cells by CRISPR-Cas9 system and detailed evaluation for clinical application

In order to expand the promise of regenerative medicine using allogeneic induced pluripotent stem cells (iPSCs), precise and efficient genome editing of human leukocyte antigen (HLA) genes would be advantageous to minimize the immune rejection caused by mismatches of HLA type. However, clinical-grade genome editing of multiple HLA genes in human iPSC lines remains unexplored. Here, we optimized the protocol for good manufacturing practice (GMP)-compatible CRISPR-Cas9 genome editing to deplete the three gene locus (HLA-A, HLA-B, and CIITA genes) simultaneously in HLA homozygous iPSCs. The use of HLA homozygous iPSCs has one main advantage over heterozygous iPSCs for inducing biallelic knockout by a single gRNA. RNA-seq and flow cytometry analyses confirmed the successful depletion of HLAs, and lineage-specific differentiation into cardiomyocytes was verified. We also confirmed that the pluripotency of genome-edited iPSCs was successfully maintained by the three germ layers of differentiation. Moreover, whole-genome sequencing, karyotyping, and optical genome mapping analyses revealed no evident genomic abnormalities detected in some clones, whereas unexpected copy number losses, chromosomal translocations, and complex genomic rearrangements were observed in other clones. Our results indicate the importance of multidimensional analyses to ensure the safety and quality of the genome-edited cells. The manufacturing and assessment pipelines presented here will be the basis for clinical-grade genome editing of iPSCs

CFD-PBM coupled simulation of a nanobubble generator with honeycomb structure

In recent years, nanobubble technologies have drawn great attention due to their wide applications in many fields of science and technology. The nitrogen nanobubble water circulation can be used to slow the progressions of oxidation and spoilage for the seafood long- term storage. From previous studies, a kind of honeycomb structure for high-efficiency nanobubble generation has been proposed. In this paper, the bubbly flow in the honeycomb structure was studied. The numerical simulations of honeycomb structure were performed by using a computational fluid dynamics–population balance model (CFD-PBM) coupled model. The numerical model was based on the Eulerian multiphase model and the population balance model (PBM) was used to calculate the gas bubble size distribution. The bubble coalescence and breakage were included. Considering the effect of bubble diameter on the fluid flow, the phase interactions were coupled with the PBM. The bubble size distributions in the honeycomb structure under different work conditions were predicted. The experimental results were compared with the simulation predictions.2018 International Conference on Material Strength and Applied Mechanics (MSAM 2018) 10–13 April 2018, Kitakyushu City, Japa

Extracellular laminin regulates hematopoietic potential of pluripotent stem cells through integrin β1-ILK-β-catenin-JUN axis

血液細胞へ効率よく変化させる弱い接着を明らかに --血液細胞の産生効率を向上--. 京都大学プレスリリース. 2021-03-31.Stuck stem cells are no good at making blood. 京都大学プレスリリース. 2021-03-31.Recombinant matrices have enabled feeder cell-free maintenance cultures of human pluripotent stem cells (hPSCs), with laminin 511-E8 fragment (LM511-E8) being widely used. However, we herein report that hPSCs maintained on LM511-E8 resist differentiating to multipotent hematopoietic progenitor cells (HPCs), unlike hPSCs maintained on LM421-E8 or LM121-E8. The latter two LM-E8s bound weakly to hPSCs compared with LM511-E8 and activated the canonical Wnt/β-catenin signaling pathway. Moreover, the extracellular LM-E8-dependent preferential hematopoiesis was associated with a higher expression of integrin β1 (ITGB1) and downstream integrin-linked protein kinase (ILK), β-catenin and phosphorylated JUN. Accordingly, the lower coating concentration of LM511-E8 or addition of a Wnt/β-catenin signaling activator, CHIR99021, facilitated higher HPC yield. In contrast, the inhibition of ILK, Wnt or JNK by inhibitors or mRNA knockdown suppressed the HPC yield. These findings suggest that extracellular laminin scaffolds modulate the hematopoietic differentiation potential of hPSCs by activating the ITGB1-ILK-β-catenin-JUN axis at the undifferentiated stage. Finally, the combination of low-concentrated LM511-E8 and a revised hPSC-sac method, which adds bFGF, SB431542 and heparin to the conventional method, enabled a higher yield of HPCs and higher rate for definitive hematopoiesis, suggesting a useful protocol for obtaining differentiated hematopoietic cells from hPSCs in general

Impulsivity and the 5-HTTLPR Polymorphism in a Non-Clinical Sample

BACKGROUND: Impulsivity has been associated with serotonergic system functions. However, few researchers have investigated the relationship between a polymorphism in the promoter of the serotonin transporter gene (5-HTTLPR) and the different components of impulsivity in a non-clinical population. The aim of this study was to investigate the relationship between a polymorphism in the promoter region of the serotonin transporter gene (5-HTTLPR) and the different components of impulsivity in a non-clinical population. METHODOLOGY/PRINCIPAL FINDINGS: We administered two neuropsychological tests, the Continuous Performance Task and the Iowa Gambling Task, to 127 healthy participants to measure their levels of motor, attentional and non-planning impulsivity. Then, these participants were grouped by genotype and gender, and their scores on impulsivity measures were compared. There were no significant differences between group scores on attentional, motor and non-planning impulsivity. CONCLUSIONS/SIGNIFICANCE: Our results suggest that 5-HTTLPR genotype is not significantly associated with subsets of impulsive behavior in a non-clinical sample when measured by neuropsychological tests. These findings are discussed in terms of the sensitivity of neuropsychological tests to detect impulsivity in a non-clinical population and the role of gender and race in the relationship between the 5-HTTLPR and impulsivity

水稲の交雑實驗中に出現したヘテロシスに就いて

The present report deals with the hybrid vigor of rice, found in a hybrid between SHINA No.2 and NANGKING SHAN-TO. Hybrid vigor occurred in some characters such as plant height, length of culm and ear, number of ears, grains per hill, diameter of culm, and size of grain. In general the growth of F_1 plants were more vigorous than the parental plants, namely the significant increases were 19.5% in culm length, 15.9% in total length of culm and ear, 16.7% in plant height, 20.0% in diameter of culm, 28.8% and 61.3% in grains per ear and hill in each other. But the increases of ear length and number of ears per hill were not significant. On the contrary, sizes of grain of F_1 was somewhat smaller as compared with that of parental plants

The Shortest Isoform of Dystrophin (Dp40) Interacts with a Group of Presynaptic Proteins to Form a Presumptive Novel Complex in the Mouse Brain

Duchenne muscular dystrophy (DMD) causes cognitive impairment in one third of the patients, although the underlying mechanisms remain to be elucidated. Recent studies showed that mutations in the distal part of the dystrophin gene correlate well with the cognitive impairment in DMD patients, which is attributed to Dp71. The study on the expression of the shortest isoform, Dp40, has not been possible due to the lack of an isoform specific antibody. Dp40 has the same promoter as that found in Dp71 and lacks the normal C-terminal end of Dp427. In the present study, we have raised polyclonal antibody against the N-terminal sequence common to short isoforms of dystrophin, including Dp40, and investigated the expression pattern of Dp40 in the mouse brain. Affinity chromatography with this antibody and the consecutive LC-MS/MS analysis on the interacting proteins revealed that Dp40 was abundantly expressed in synaptic vesicles and interacted with a group of presynaptic proteins, including syntaxin1A and SNAP25, which are involved in exocytosis of synaptic vesicles in neurons. We thus suggest that Dp40 may form a novel protein complex and play a crucial role in presynaptic function. Further studies on these aspects of Dp40 function might provide more insight into the molecular mechanisms of cognitive impairment found in patients with DMD

Derivation, simulation and validation of a cohesive particle flow CFD model

A comprehensive physical model describing the agglomeration behavior present during fluidization of fine powders is still missing in literature. In this work, a model of balance of forces acting on a single solid particle is introduced, aiming at predicting and locally estimating the size of the agglomerates created in the bed. Computational Fluid Dynamics (CFD) have been used to investigate the hydrodynamics of a gas-solid fluidized bed operated with particles belonging to group A of Geldart classification(Geldart, 1973). The key issue is that, in the gas and particle flow field, both hydrodynamic and inter-particle forces are of importance. The model is incorporated into simulations based on an Eulerian approach and using the kinetic theory of granular flow. In the simulations, the closure models describing the hydrodynamics of the solids phase are directly affected by the behavior of the agglomerates. No empirical data or parameters were used to close the model. The simulations are compared with experiments of an independent research group, through the time-averaged solids volume fraction in a fluidized bed operated at different gas velocities. The agreement obtained between the simulation results and data from the literature is very good. Also, it is shown that, under flow conditions treated in the present work, agglomerates of size of several single particle diameters are present in the fluidized bed

水稲の栽培時期の早晩と胚乳内澱粉の粘度との関係

水稲の栽培時期の早晩と, 収穫された玄米の胚乳内澱粉の粘度との関係を検討した。実験は1954及び1956年の両年度に粳稲2品種と糯稲3品種とを供試して行われた。粘度測定にはOstward粘度計を用いた。主な実験結果は次の通りである。i) 糯品種の胚乳内澱粉の比粘度は粳品種のそれにくらべて高く, また品種間の差異として, 所謂日本型に属する品種では印度型品種よりも例外なく高い値を示した。ii) 胚乳内澱粉の比粘度は, 当地方の慣行播種期(5月中旬)に近く播種された区に於て, 全品種共常に最高の値を示した。それよりも早く, また晩くに栽培されたものでは低くなり特に過度に晩播晩植した場合にはこの傾向が顯著である。iii) 胚乳内澱粉の比粘度と出穗後成熟に至る期間の積算温度との間には負の相関々係があり, 特に日本型の品種では明かに有意の相関係数を示す。また同じ期間の日温度較差の平均値と比粘度との間にも負の相関々係の存在がうかゞわれるが, 1品種を除いては相関係数は有意ではない。iv) これらの結果からして, 胚乳内澱粉の粘度は出穗後の種々な温度条件によつてかなり左右され, またごく一般的にいうならば, 他の研究者達も述べているように, この期間の気候条件が米粒中の炭水化物の形成や集積に複雑な関連を示すことを裏づけるものと思われる。In this experiment, conducted in 1954,and 1956,changes in starch viscosity of the endosperm were examined by the authors, with five varieties including glutinous and non-glutinous. They were sown at four different periods and then normaly cultivated, that is, four different cultivations, early or late, were carried out. Viscosity (ηsp) was estimated by the help of the Ostward viscositymeter with starch elaborated from grains after the removal of the embryo. Main results were as those : 1) In all glutinous varieties, the starch showed higher viscosity than non-glutinous ones. Moroover, varietal differences were observed between the so-called Japonica and Indica types. The varieties belonging to the former type have always higher values in viscosity than the latters. 2) In the case of the planting seeded at May 20 in all varieties, starch viscosity showed the highest values as compared with those plantings sown earlier or later than this. Particularly when they were seeded most lately, the viscosity was reduced strikingly (Table 1 & Fig. 1). 3) Starch viscosity was found to be nagatively correlated with the sum of temperatures during the period from heading to ripening. Especially it was conspicuous among Japanese varieties as presented in Table 2 & Fig. 2. With the exception of only one variety (Akamoti), the negative correlation between viscosity and the average amplitudes of temperature was never recognizable. 4) From these facts mentioned above, starch viscosity of the endosperm seemed to be affected by temperature conditions after heading. In general, the results obtained may support the observations of other workers showing the complicated relationship between synthetic accumulation of carbohydrates in rice grain and climatic factors during the ripening period

糯稲の類型化に関する考察(農学部門)

糯稲品種が, その胚乳澱粉粘度と玄米の緑化時水分含量を指標とする「糯性程度」(grade of glutinous nature)の差によつて分類されることを提案した。内・外国産26品種をこの方法によつて5つの類型に大別した。「糯性程度」の高い品種程, 一般に多蘗性で籾藁比率も高い。また「糯性程度」の最も高い品種は草丈や穂長が短く, 逆に最も低い品種は長稈, 長穂の傾向を示した。中間には, これらの形質について雑多な品種が混在している。日本及び朝鮮産品種あるいはA型品種群は, 比較的「糯性程度」の高いIII, IV, V型に属するものが多く, これに対して外国産あるいはB, C型品種は「糯性程度」と早晩性との間には明瞭な関係はないが, I型品種は例外なく晩生種である。糯稲にみられるこのような性質の相違は, 各種の生態的条件や民族の嗜好性による選抜の結果として分化したものと考えられる。(1) The authors tried to classify glutinous rice plants by their "grade of glutinous nature", using 26 varieties, 13 of Japan, 5 of China, 2 of Korea, 5 of Southeast Asia, 1 of Italy and 1 of South America (Table 1). Five grades such as I∿V were proposed by combining both starch viscosity of endosperm and water contents of grain at the time Ryokka happend, of which the most important properties in glutinous rice plant. From grade I to V, viscosity and water contents of grain became higher in each value as presented in Table 2 & Fig. 1. (2) Number of ears and ratio of grain to straw, in general, went hand in hand with the grade, showing larger values. Both culm and ear of varieties belonged the highest grade were shorter in length and vice versa in the case of varieties belonged the lowest one (Fig. 2). (3) Majority of varieties native to Japan and Korea, or A type grouped by MATSUO (1952), belonged to grade III, IV and V, while varieties distributed in Southeast Asia, or B and C types included large number of grade I and II. The correlation between the grade and date of heading was never recognizable, but it was found that varieties belonged grade I headed later with no exception (Fig. 3). (4) The differentiation of "grade of glutinous nature" in rice varieties as pointed above was thought to had been caused by selection of different environment and different taste of people