FAST-id system for enrichment of cells with TALEN-induced mutations and large deletions.

Transcription activator-like effector nuclease (TALEN)-mediated genome editing is a powerful technique for analyzing gene functions in various cells and organisms. At target loci, TALENs can not only introduce short insertions and deletions, but also yield large deletions through the use of two TALEN pairs. Here, we report easy and efficient methods for enrichment of cells with TALEN-induced mutations and large deletions. First, we established the fluorescence-activated sorting of TALEN-induced deletions (FAST-id) system that enabled fluorescence-activated cell sorting-mediated enrichment of cells with TALEN-induced mutations. In the FAST-id system, either EGFP or mCherry and TALENs were co-expressed. Using dual fluorescence selection, both left and right TALEN-expressing cells were easily concentrated, resulting in enrichment of TALEN-mediated mutated cells. Next, to apply the FAST-id system to enrichment of cells with large deletions, we developed the fast unification of separate endonucleases (FUSE) method for assembly of two TALENs into a single expression vector. Using the FUSE method, we easily obtained a TALEN pair-expressing plasmid driven by a single promoter. By combining the FAST-id system and FUSE method, cells with large deletions were efficiently enriched. To the best of our knowledge, this is the first report of enrichment of cells with TALEN-induced large deletions.

Efficient TALEN construction and evaluation methods for human cell and animal applications.

Transcription activator-like effector nucleases (TALENs) have recently arisen as effective tools for targeted genome engineering. Here, we report streamlined methods for the construction and evaluation of TALENs based on the 'Golden Gate TALEN and TAL Effector Kit' (Addgene). We diminished array vector requirements and increased assembly rates using six-module concatemerization. We altered the architecture of the native TALEN protein to increase nuclease activity and replaced the final destination vector with a mammalian expression/in vitro transcription vector bearing both CMV and T7 promoters. Using our methods, the whole process, from initiating construction to completing evaluation directly in mammalian cells, requires only 1 week. Furthermore, TALENs constructed in this manner may be directly applied to transfection of cultured cells or mRNA synthesis for use in animals and embryos. In this article, we show genomic modification of HEK293T cells, human induced pluripotent stem cells, Drosophila melanogaster, Danio rerio and Xenopus laevis, using custom-made TALENs constructed and evaluated with our protocol. Our methods are more time efficient compared with conventional yeast-based evaluation methods and provide a more accessible and effective protocol for the application of TALENs in various model organisms.

Targeted mutagenesis in sea urchin embryos using TALENs.

Genome editing with engineered nucleases such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) has been reported in various animals. We previously described ZFN-mediated targeted mutagenesis and insertion of reporter genes in sea urchin embryos. In this study, we demonstrate that TALENs can induce mutagenesis at specific genomic loci of sea urchin embryos. Injection of TALEN mRNAs targeting the HpEts transcription factor into fertilized eggs resulted in the impairment of skeletogenesis. Sequence analyses of the mutations showed that deletions and/or insertions occurred at the HpEts target site in the TALEN mRNAs-injected embryos. The results suggest that targeted gene disruption using TALENs is feasible in sea urchin embryos.

Nurses' Attitudes, Practices, and Barriers to Assessing Symptoms of Discomfort in Mechanically Ventilated Patients: A Cross-Sectional Study.

Introduction: Critically ill patients experience various stressful symptoms of discomfort, including dyspnea, pain, and sleep disruption. Notably, ventilated patients have difficulty self-reporting discomfort symptoms. Nurses need to assess discomfort symptoms to alleviate them, but limited research exists on discomfort symptom assessment and management in critically ill patients. Objective: To identify the practices, attitudes, and barriers among nurses related to the assessment of discomfort symptoms in mechanically ventilated patients. Methods: Using a cross-sectional, descriptive study design, a web-based survey was conducted between May and June 2022 with critical care nurses sampled through Japanese academic societies and social networking services. The survey contained questions relative to the above-stated objective. Descriptive statistical analysis was performed without sample size calculation because of the descriptive and exploratory nature of this study. Results: There were 267 respondents to the questionnaire. The discomfort symptoms that nurses perceived as important to assess were pain (median 100 [interquartile range, IQR 90-100]), insomnia (99 [80-100]), and dyspnea (96.5 [75-100]). Most participants (89.8%) routinely assessed pain in mechanically ventilated patients using a scale; however, other discomfort symptoms were assessed by less than 40% (dyspnea [28.4%], fatigue [8.1%], thirst [13.1%], insomnia [37.3%], and anxiety [13.6%]). Two major barriers to assessing discomfort symptoms were lack of assessment culture within the intensive care unit and lack of knowledge of the relevant evaluation scales. Conclusions: Nurses were aware of the importance of using scales to assess the discomfort symptoms experienced by mechanically ventilated patients. However, except for pain, most nurses did not routinely use scales to assess discomfort symptoms. Barriers to routine discomfort symptom assessment included the lack of an assessment culture and the lack of knowledge of the assessment scales. Clinicians should be educated regarding the existence of validated rating scales and develop additional rating scales utilizable for minor discomforts in mechanically ventilated patients.

Effect of sequencing platforms on the sensitivity of chemical mutation detection using Hawk-Seq™.

BACKGROUND: Error-corrected next-generation sequencing (ecNGS) technologies have enabled the direct evaluation of genome-wide mutations after exposure to mutagens. Previously, we reported an ecNGS methodology, Hawk-Seq™, and demonstrated its utility in evaluating mutagenicity. The evaluation of technical transferability is essential to further evaluate the reliability of ecNGS-based assays. However, cutting-edge sequencing platforms are continually evolving, which can affect the sensitivity of ecNGS. Therefore, the effect of differences in sequencing instruments on mutation data quality should be evaluated. RESULTS: We assessed the performance of four sequencing platforms (HiSeq2500, NovaSeq6000, NextSeq2000, and DNBSEQ-G400) with the Hawk-Seq™ protocol for mutagenicity evaluation using DNA samples from mouse bone marrow exposed to benzo[a]pyrene (BP). The overall mutation (OM) frequencies per 106 bp in vehicle-treated samples were 0.22, 0.36, 0.46, and 0.26 for HiSeq2500, NovaSeq6000, NextSeq2000, and DNBSEQ-G400, respectively. The OM frequency of NextSeq2000 was significantly higher than that of HiSeq2500, suggesting the difference to be based on the platform. The relatively higher value in NextSeq2000 was a consequence of the G:C to C:G mutations in NextSeq2000 data (0.67 per 106 G:C bp), which was higher than the mean of the four platforms by a ca. of 0.25 per 106 G:C bp. A clear dose-dependent increase in G:C to T:A mutation frequencies was observed in all four sequencing platforms after BP exposure. The cosine similarity values of the 96-dimensional trinucleotide mutation patterns between HiSeq and the three other platforms were 0.93, 0.95, and 0.92 for NovaSeq, NextSeq, and DNBSeq, respectively. These results suggest that all platforms can provide equivalent data that reflect the characteristics of the mutagens. CONCLUSIONS: All platforms sensitively detected mutagen-induced mutations using the Hawk-Seq™ analysis. The substitution types and frequencies of the background errors differed depending on the platform. The effects of sequencing platforms on mutagenicity evaluation should be assessed before experimentation.

Grip Strength Correlates with Mental Health and Quality of Life after Critical Care: A Retrospective Study in a Post-Intensive Care Syndrome Clinic.

Post-intensive care syndrome (PICS) is characterized by several prolonged symptoms after critical care, including physical and cognitive dysfunctions as well as mental illness. In clinical practice, the long-term follow-up of PICS is initiated after patients have been discharged from the intensive care unit, and one of the approaches used is a PICS clinic. Although physical dysfunction and mental illness often present in combination, they have not yet been examined in detail in PICS patients. Grip strength is a useful physical examination for PICS, and is reported to be associated with mental status in the elderly. We herein investigated the relationship between grip strength and the mental status using data from our PICS clinic. We primarily aimed to analyze the correlation between grip strength and the Hospital Anxiety and Depression Scale (HADS) score. We also analyzed the association between grip strength and the EuroQol 5 Dimension (EQ5D) score as quality of life (QOL). Subjects comprised 133 patients who visited the PICS clinic at one month after hospital discharge between August 2019 and December 2020. Total HADS scores were 7 (4, 13) and 10 (6, 16) (p = 0.029) and EQ5D scores were 0.96 (0.84, 1) and 0.77 (0.62, 0.89) (p ≤ 0.0001) in the no walking disability group and walking disability group, respectively. Grip strength negatively correlated with HADS and EQ5D scores. Correlation coefficients were r = -0.25 (p = 0.011) and r = -0.47 (p < 0.0001) for HADS and EQ5D scores, respectively. Grip strength was a useful evaluation that also reflected the mental status and QOL.

High-fat diet impairs cognitive function of zebrafish.

An unhealthy diet with excessive fat intake has often been claimed to induce not only obesity but also cognitive dysfunction in mammals; however, it is not known whether this is the case in zebrafish. Here, we investigated the effect of excessive fat in the diet on cognitive function and on gene expression in the telencephalon of zebrafish. Cognitive function, as measured by active avoidance test, was impaired by feeding of a high-fat diet compared with a control diet. In RNA sequencing analysis of the telencephalon, 97 genes were identified with a fold change in expression greater than 2 and a p-value less than 0.05 between the two diets. In quantitative real-time PCR analysis of the telencephalon, genes related to neuronal activity, anti-oxidative stress, blood-brain barrier function and amyloid-ß degradation were found to be downregulated, whereas genes related to apoptosis and amyloid-ß production were found to be upregulated, in the high-fat diet group, which are changes known to occur in mammals fed a high-fat diet. Collectively, these results are similar to those found in mammals, suggesting that zebrafish can serve as a suitable animal model in research into cognitive impairment induced by excessive fat in the diet.