One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT)

Ohtsuka, M., Miura, H., Mochida, K. et al. One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT). BMC Genomics 16, 274 (2015). https://doi.org/10.1186/s12864-015-1432-

Assessing Burden, Anxiety, Depression, and Quality of Life among Caregivers of Hemodialysis Patients in Indonesia: A Cross-Sectional Study

Family caregivers endure the burden of caring for patients receiving hemodialysis, which can affect their psychological status and may disrupt the care process. This study aims to assess the level of burden, anxiety, depression, and quality of life (QOL) among family caregivers, to investigate the influence of caregivers’ sociodemographic factors and patients’ clinical conditions on the level of burden, and investigate how burden affects anxiety/depression and QOL. A descriptive, cross-sectional study was conducted from September to October 2020. A total of 104 caregivers with a mean age of 44.4 ± 12.7 years (63.5% women) in the hemodialysis department of a hospital in Indonesia were examined. Zarit Burden Interview (ZBI), Hospital Anxiety and Depression Scale, and WHOQOL-BREF were used. Descriptive analysis was conducted to assess the level of psychological status, and multiple regression analysis and path analysis were performed to evaluate the association among all factors. As result, regarding burden, 10.2% had a moderate-to-severe burden, and in terms of anxiety and depression, 25% and 9.6% showed abnormal cases; the mean scores of 4 domains of QOL were about 60 points. Burden and anxiety/depression were significantly negatively associated with all domains of QOL (p < 0.001); however, sociodemographic and clinical factors were not associated with any of the QOL domains. The path analysis results showed that burden positively correlated with anxiety/depression and negatively correlated with QOL via anxiety/depression. In conclusion, family caregivers’ QOL was found to be indirectly influenced by burden through anxiety/depression. Further evaluation of clinical significance and implications for caregiver’s lifestyle regarding anxiety and depression, which result in caregiver burden, is warranted

コロンブス ノ タマゴ テキ ハッソウ ニ モトズイタ タイイ ヘンカンヨウ マット ノ シサク

We report on a new mat experimentally developed by us for changing the position of the body (named i-mat), based on an idea of a floating body. [Method] At first,4, approximately 70cm long, belts were attached to both the right and left side of the pad for tying to the beds. For a lateral position with a 30-degree tilt to the right side, the belt attached on the left side is fixed to the right bed fence and the length of the belt is adjusted so that the patient’s position becomes lateral with a 30-degree tilt. The body pressure levels (mmHg) at sites of bone were measured after position changes by using a simple body pressure-measuring device (PREDIA®) in 10 patients with an independence degree of daily living rating of C2 (group i) in whom the i-mat was used, and compared with the levels measured after position changes in patients where a mat made of urethane (Nasentpat®) was used (group N). [Results] The pressure levels in the sacral region and occipital region could be significantly lowered in group i as compared with that in group N. We also measured and compared the pressures at the major trochanter, acrominon, and iliac regions, because the high-pressure loaded region shifted to these regions on the side facing the bed in the lateral position. No significant differences in the pressure levels at the major trochanter and acrominon were found between the i and N groups. The pressure level in the iliac region in the i group was significantly higher than that that in the N group. [Conclusion] Use of this experimentally produced i-mat can significantly lower the pressure load at the sacral and occipital regions when the patient is placed in the lateral position of either side with a 30-degree tilt, as compared to that of Nasentpat®. But it appears that caution should be exercised to prevent the occurrence of bedsores in the iliac region

Molecular Identification of t(w5): Vps52 Promotes Pluripotential Cell Differentiation Through Cell-Cell Interactions

After implantation, pluripotent epiblasts are converted to embryonic ectoderm through cell-cell interactions that significantly change the transcriptional and epigenetic networks. An entree to understanding this vital developmental transition is the t(w5) mutation of the mouse t complex. This mutation produces highly specific defects in the embryonic ectoderm before gastrulation, leading to death of the embryonic ectoderm. Using a positional cloning approach, we have now identified the mutated gene, completing a decades-long search. The gene, vacuolar protein sorting 52 (Vps52), is a mouse homolog of yeast VPS52 that is involved in the retrograde trafficking of endosomes. Our data suggest that Vps52 acts in extraembryonic tissues to support the growth and differentiation of embryonic ectoderm via cell-cell interactions. It is also required in the formation of embryonic structures at a later stage of development, revealing hitherto unknown functions of Vps52 in the development of a multicellular organism.NSFCellular and Molecular Biolog

Mechanistic insights into intramembrane proteolysis by E. coli site-2 protease homolog RseP

細胞膜の中ではたらく特殊なタンパク質分解酵素の構造を解明 --細菌感染症の新たな治療法の開発へ期待--. 京都大学プレスリリース. 2022-08-25.Site-2 proteases are a conserved family of intramembrane proteases that cleave transmembrane substrates to regulate signal transduction and maintain proteostasis. Here, we elucidated crystal structures of inhibitor-bound forms of bacterial site-2 proteases including Escherichia coli RseP. Structure-based chemical modification and cross-linking experiments indicated that the RseP domains surrounding the active center undergo conformational changes to expose the substrate-binding site, suggesting that RseP has a gating mechanism to regulate substrate entry. Furthermore, mutational analysis suggests that a conserved electrostatic linkage between the transmembrane and peripheral membrane-associated domains mediates the conformational changes. In vivo cleavage assays also support that the substrate transmembrane helix is unwound by strand addition to the intramembrane β sheet of RseP and is clamped by a conserved asparagine residue at the active center for efficient cleavage. This mechanism underlying the substrate binding, i.e., unwinding and clamping, appears common across distinct families of intramembrane proteases that cleave transmembrane segments

Discrimination of Stem Cell Status after Subjecting Cynomolgus Monkey Pluripotent Stem Cells to Naïve Conversion

Experimental animal models have played an indispensable role in the development of human induced pluripotent stem cell (iPSC) research. The derivation of high-quality (so-called “true naïve state”) iPSCs of non-human primates enhances their application and safety for human regenerative medicine. Although several attempts have been made to convert human and non-human primate PSCs into a truly naïve state, it is unclear which evaluation methods can discriminate them as being truly naïve. Here we attempted to derive naïve cynomolgus monkey (Cm) (Macaca fascicularis) embryonic stem cells (ESCs) and iPSCs. Several characteristics of naïve Cm ESCs including colony morphology, appearance of naïve-related mRNAs and proteins, leukaemia inhibitory factor dependency, and mitochondrial respiration were confirmed. Next, we generated Cm iPSCs and converted them to a naïve state. Transcriptomic comparison of PSCs with early Cm embryos elucidated the partial achievement (termed naïve-like) of their conversion. When these were subjected to in vitro neural differentiation, enhanced differentiating capacities were observed after naïve-like conversion, but some lines exhibited heterogeneity. The difficulty of achieving contribution to chimeric mouse embryos was also demonstrated. These results suggest that Cm PSCs could ameliorate their in vitro neural differentiation potential even though they could not display true naïve characteristics

A High-Speed Congenic Strategy Using First-Wave Male Germ Cells

BACKGROUND: In laboratory mice and rats, congenic breeding is essential for analyzing the genes of interest on specific genetic backgrounds and for analyzing quantitative trait loci. However, in theory it takes about 3-4 years to achieve a strain carrying about 99% of the recipient genome at the tenth backcrossing (N10). Even with marker-assisted selection, the so-called 'speed congenic strategy', it takes more than a year at N4 or N5. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a new high-speed congenic system using round spermatids retrieved from immature males (22-25 days of age). We applied the technique to three genetically modified strains of mice: transgenic (TG), knockin (KI) and N-ethyl-N-nitrosourea (ENU)-induced mutants. The donor mice had mixed genetic backgrounds of C57BL/6 (B6):DBA/2 or B6:129 strains. At each generation, males used for backcrossing were selected based on polymorphic marker analysis and their round spermatids were injected into B6 strain oocytes. Backcrossing was repeated until N4 or N5. For the TG and ENU-mutant strains, the N5 generation was achieved on days 188 and 190 and the proportion of B6-homozygous loci was 100% (74 markers) and 97.7% (172/176 markers), respectively. For the KI strain, N4 was achieved on day 151, all the 86 markers being B6-homozygous as early as on day 106 at N3. The carrier males at the final generation were all fertile and propagated the modified genes. Thus, three congenic strains were established through rapid generation turnover between 41 and 44 days. CONCLUSIONS/SIGNIFICANCE: This new high-speed breeding strategy enables us to produce congenic strains within about half a year. It should provide the fastest protocol for precise definition of the phenotypic effects of genes of interest on desired genetic backgrounds