A universal method for generating knockout mice in multiple genetic backgrounds using zygote electroporation.

Genetically engineered mouse models are essential tools for understanding mammalian gene functions and disease pathogenesis. Genome editing allows the generation of these models in multiple inbred strains of mice without backcrossing. Zygote electroporation dramatically removed the barrier for introducing the CRISPR-Cas9 complex in terms of cost and labour. Here, we demonstrate that the generalised zygote electroporation method is also effective for generating knockout mice in multiple inbred strains. By combining in vitro fertilisation and electroporation, we obtained founders for knockout alleles in eight common inbred strains. Long-read sequencing analysis detected not only intended mutant alleles but also differences in read frequency of intended and unintended alleles among strains. Successful germline transmission of knockout alleles demonstrated that our approach can establish mutant mice targeting the same locus in multiple inbred strains for phenotyping analysis, contributing to reverse genetics and human disease research.

Growth acceleration of Nile tilapia at 21 to 31 weeks of age with plasma-treated air-supplied water.

This study aimed to develop a technique to accelerate fish growth without using genetic modification or genome editing. We have prepared a reactor with four pairs of opposed electrodes and a high-voltage power supply for the discharge. An arc discharge generates a plasma-treated gas in the reactor. Plasma-treated gas containing active species such as nitric oxide (NO) was generated via an arc discharge in the atmosphere and inserted into an aquarium containing Nile tilapia. No ozone was detected in the plasma-treated gas. Plasma treatment gas was supplied to the 20 L tank at a flow rate of 10 L per minute for varying supply times. The supply duration of plasma-treated air to the water tank was 0.5, 2, 5, and 15 min. Tanks were prepared for each of these four conditions, and gas was supplied daily at the same time. We observed that on supplying plasma-treated gas to tilapia from the 16th week of age for 5 min daily, the average length of the fish at 31 weeks of age was ∼1.5 times longer than that of the control fish. All other supply time conditions were also found to grow acceleration over the control. In the 15-minute supply time condition, individual differences in body length were more significant. A sample had more growth suppression than controls. In other words, the results suggest that an excess supply of active species can cause growth inhibition. These results suggest that an optimal supply of plasma-treated gas has a growth-promoting effect on fish.Key policy highlightsThe fish growth was accelerated by supplying plasma-treated air to the tank.The amount of ozone in the plasma-treated air was below the detection limit, and a large amount of RNS, such as nitric oxide, was generated.After an experimental period of 16 to 31 weeks, the average length of fish in the most significant growth condition was 1.5 times that of the control fish.

DAJIN enables multiplex genotyping to simultaneously validate intended and unintended target genome editing outcomes.

Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

MEG activity of the dorsolateral prefrontal cortex during optic flow stimulations detects mild cognitive impairment due to Alzheimer's disease.

Dorsal stream, which has a neuronal connection with dorsolateral prefrontal cortex (DLPFC), is known to be responsible for detection of motion including optic flow perception. Using magnetoencephalography (MEG), this study aimed to examine neural responses to optic flow stimuli with looming motion in the DLPFC in patients with mild cognitive impairment due to Alzheimer's disease (AD-MCI) compared with cognitively unimpaired participants (CU). We analyzed the neural responses by evaluating maximum source-localized power for the AD-MCI group (n = 11) and CU (n = 20), focusing on six regions of interest (ROIs) that form the DLPFC: right and left dorsal Brodmann area 9/46 (A9/46d), Brodmann area 46 (A46) and ventral Brodmann area 9/46 (A9/46v). We found significant differences in the maximum power between the groups in the left A46 and A9/46v. Moreover, in the left A9/46v, the maximum power significantly correlated with the Wechsler Memory Scale-Revised general memory score and delayed recall score. The maximum power in the left A9/46v also revealed high performance in AD-MCI versus CU classification with the area under the ROC curve of 0.90. This study demonstrated that MEG during the optic flow task can be useful in discriminating AD-MCI from CU.

Post-Embryonic Lateral Organ Development and Adaxial-Abaxial Polarity Are Regulated by the Combined Effect of ENHANCER OF SHOOT REGENERATION 1 and WUSCHEL in Arabidopsis Shoots.

The development of above-ground lateral organs is initiated at the peripheral zone of the shoot apical meristem (SAM). The coordination of cell fate determination and the maintenance of stem cells are achieved through a complex regulatory network comprised of transcription factors. Two AP2/ERF transcription factor family genes, ESR1/DRN and ESR2/DRNL/SOB/BOL, regulate cotyledon and flower formation and de novo organogenesis in tissue culture. However, their roles in post-embryonic lateral organ development remain elusive. In this study, we analyzed the genetic interactions among SAM-related genes, WUS and STM, two ESR genes, and one of the HD-ZIP III members, REV, whose protein product interacts with ESR1 in planta. We found that esr1 mutations substantially enhanced the wus and stm phenotypes, which bear a striking resemblance to those of the wus rev and stm rev double mutants, respectively. Aberrant adaxial-abaxial polarity is observed in wus esr1 at relatively low penetrance. On the contrary, the esr2 mutation partially suppressed stm phenotypes in the later vegetative phase. Such complex genetic interactions appear to be attributed to the distinct expression pattern of two ESR genes because the ESR1 promoter-driving ESR2 is capable of rescuing phenotypes caused by the esr1 mutation. Our results pose the unique genetic relevance of ESR1 and the SAM-related gene interactions in the development of rosette leaves.

EXOC1 plays an integral role in spermatogonia pseudopod elongation and spermatocyte stable syncytium formation in mice.

The male germ cells must adopt the correct morphology at each differentiation stage for proper spermatogenesis. The spermatogonia regulates its differentiation state by its own migration. The male germ cells differentiate and mature with the formation of syncytia, failure of forming the appropriate syncytia results in the arrest at the spermatocyte stage. However, the detailed molecular mechanisms of male germ cell morphological regulation are unknown. Here, we found that EXOC1, a member of the Exocyst complex, is important for the pseudopod formation of spermatogonia and spermatocyte syncytia in mice. EXOC1 contributes to the pseudopod formation of spermatogonia by inactivating the Rho family small GTPase Rac1 and also functions in the spermatocyte syncytia with the SNARE proteins STX2 and SNAP23. Since EXOC1 is known to bind to several cell morphogenesis factors, this study is expected to be the starting point for the discovery of many morphological regulators of male germ cells.

Interpreting Cytokinin Action as Anterograde Signaling and Beyond.

Among the major phytohormones, the cytokinin exhibits unique features for its ability to positively affect the developmental status of plastids. Even early on in its research, cytokinins were known to promote plastid differentiation and to reduce the loss of chlorophyll in detached leaves. Since the discovery of the components of cytokinin perception and primary signaling, the genes involved in photosynthesis and plastid differentiation have been identified as those directly targeted by type-B response regulators. Furthermore, cytokinins are known to modulate versatile cellular processes such as promoting the division and differentiation of cells and, in concert with auxin, initiating the de novo formation of shoot apical meristem (SAM) in tissue cultures. Yet how cytokinins precisely participate in such diverse cellular phenomena, and how the associated cellular processes are coordinated as a whole, remains unclear. A plausible presumption that would account for the coordinated gene expression is the tight and reciprocal communication between the nucleus and plastid. The fact that cytokinins affect plastid developmental status via gene expression in both the nucleus and plastid is interpreted here to suggest that cytokinin functions as an initiator of anterograde (nucleus-to-plastid) signaling. Based on this viewpoint, we first summarize the physiological relevance of cytokinins to the coordination of plastid differentiation with de novo shoot organogenesis in tissue culture systems. Next, the role of endogenous cytokinins in influencing plastid differentiation within the SAM of intact plants is discussed. Finally, a presumed plastid-derived signal in response to cytokinins for coupled nuclear gene expression is proposed.

Clarification of electrical current importance in plasma gene transfection by equivalent circuit analysis.

We have been developing a method of plasma gene transfection that uses microdischarge plasma (MDP) and is highly efficient, minimally invasive, and safe. Using this technique, electrical factors (such as the electrical current and electric field created through processing discharge plasma) and the chemical factors of active species and other substances focusing on radicals are supplied to the cells and then collectively work to introduce nucleic acids in the cell. In this paper, we focus on the electrical factors to identify whether the electric field or electrical current is the major factor acting on the cells. More specifically, we built a spatial distribution model that uses an electrical network to represent the buffer solution and cells separately, as a substitute for the previously reported uniform medium model (based on the finite element method), calculated the voltage and electrical current acting on cells, and examined their intensity. Although equivalent circuit models of single cells are widely used, this study was a novel attempt to build a model wherein adherent cells distributed in two dimensions were represented as a group of equivalent cell circuits and analyzed as an electrical network that included a buffer solution and a 96-well plate. Using this model, we could demonstrate the feasibility of applying equivalent circuit network analysis to calculate electrical factors using fewer components than those required for the finite element method, with regard to electrical processing systems targeting organisms. The results obtained through this equivalent circuit network analysis revealed for the first time that the distribution of voltage and current applied to a cellular membrane matched the spatial distribution of experimentally determined gene transfection efficiency and that the electrical current is the major factor contributing to introduction.

Efficient production of large deletion and gene fragment knock-in mice mediated by genome editing with Cas9-mouse Cdt1 in mouse zygotes.

Genetically modified mouse models are essential for in vivo investigation of gene function and human disease research. Targeted mutations can be introduced into mouse embryos using genome editing technology such as CRISPR-Cas. Although mice with small indel mutations can be produced, the production of mice carrying large deletions or gene fragment knock-in alleles remains inefficient. We introduced the nuclear localisation property of Cdt1 protein into the CRISPR-Cas system for efficient production of genetically engineered mice. Mouse Cdt1-connected Cas9 (Cas9-mC) was present in the nucleus of HEK293T cells and mouse embryos. Cas9-mC induced a bi-allelic full deletion of Dmd, GC-rich fragment knock-in, and floxed allele knock-in with high efficiency compared to standard Cas9. These results indicate that Cas9-mC is a useful tool for producing mouse models carrying targeted mutations.

First Come, First Served: Sui Generis Features of the First Intron.

Most of the transcribed genes in eukaryotic cells are interrupted by intervening sequences called introns that are co-transcriptionally removed from nascent messenger RNA through the process of splicing. In Arabidopsis, 79% of genes contain introns and more than 60% of intron-containing genes undergo alternative splicing (AS), which ostensibly is considered to increase protein diversity as one of the intrinsic mechanisms for fitness to the varying environment or the internal developmental program. In addition, recent findings have prevailed in terms of overlooked intron functions. Here, we review recent progress in the underlying mechanisms of intron function, in particular by focusing on unique features of the first intron that is located in close proximity to the transcription start site. The distinct deposition of epigenetic marks and nucleosome density on the first intronic DNA sequence, the impact of the first intron on determining the transcription start site and elongation of its own expression (called intron-mediated enhancement, IME), translation control in 5'-UTR, and the new mechanism of the trans-acting function of the first intron in regulating gene expression at the post-transcriptional level are summarized.

Spontaneous MEG activity of the cerebral cortex during eyes closed and open discriminates Alzheimer's disease from cognitively normal older adults.

This study aimed to examine whether magnetoencephalography (MEG) is useful to detect early stage Alzheimer's disease (AD). We analyzed MEG data from the early stage AD group (n = 20; 6 with mild cognitive impairment due to AD and 14 with AD dementia) and cognitively normal control group (NC, n = 27). MEG was recorded during resting eyes closed (EC) and eyes open (EO), and the following 6 values for each of 5 bands (θ1: 4-6, θ2: 6-8, α1: 8-10, α2: 10-13, ß: 13-20 Hz) in the cerebral 68 regions were compared between the groups: (1) absolute power during EC and (2) EO, (3) whole cerebral normalization (WCN) power during EC and (4) EO, (5) difference of the absolute powers between the EC and EO conditions (the EC-EO difference), and (6) WCN value of the EC-EO difference. We found significant differences between the groups in the WCN powers during the EO condition, and the EC-EO differences. Using a Support Vector Machine classifier, a discrimination accuracy of 83% was obtained and an AUC in an ROC analysis was 0.91. This study demonstrates that MEG during resting EC and EO is useful in discriminating between early stage AD and NC.

Higher Blood Vitamin C Levels are Associated with Reduction of Apolipoprotein E E4-related Risks of Cognitive Decline in Women: The Nakajima Study.

BACKGROUND: Antioxidants like vitamins C and E may minimize the risk for Alzheimer's disease. OBJECTIVE: We examined whether vitamins C and E modify the apolipoprotein E (APOE) E4-related risks for developing cognitive decline. METHODS: We conducted a population-based prospective study including Japanese residents aged 65 years from Nakajima, Japan. The participants received an evaluation of cognitive function and underwent blood tests including tests for vitamins C and E levels and APOE phenotypes. The APOE E4-by-gender-by-vitamin C or E interactions on developing cognitive decline were analyzed. RESULTS: Of 606 participants with normal cognitive function determined using a baseline survey (2007-2008), 349 completed the follow up survey between 2014 and 2016. In women with APOE E4, significantly reduced risk for cognitive decline was observed for the highest blood vitamin C concentration tertile [multivariate OR 0.10 (95% CI 0.01-0.93)] compared with the lowest tertile. In men without APOE E4, significantly reduced risk for cognitive decline was observed for the highest blood vitamin E concentration tertile [multivariate OR 0.19 (0.05-0.74)] as compared with the lowest tertile. CONCLUSION: Our results demonstrate significant beneficial effects of vitamins C and E in reducing the risk of cognitive decline in women with APOE E4 and men without APOE E4, respectively.

Differences in Dementia Beliefs between Non-Demented Public Screeners and In-Home Screeners and Their Potential Impact on Future Dementia Screening Intention: The Nakajima Study.

In many cohort studies of dementia, while differences in sociodemographic characters between responders and non-responders of dementia screening have been reported, differences in dementia beliefs have been relatively less known. The aims of this study were to clarify dementia beliefs and to explore potential impacts on an intention to attend a future dementia screening in public screeners and in-home screeners, respectively. We performed a cross-sectional population-based study using a question about an intention to attend a future dementia screening and a questionnaire on dementia beliefs. Subjects were all residents aged 65 years or older in the north area of Nakajima, Japan (n = 385). All subjects were asked to attend a public dementia screening first. An in-home dementia screening was subsequently conducted in subjects with non-responders to a public screening. The questionnaire consisted of four dementia beliefs: "perceived susceptibility," "perceived severity," "perceived barriers," and "perceived benefits." Public screeners significantly expressed an intention to attend a future dementia screening more than in-home screeners (p = 0.002). In in-home screeners, low "perceived severity" were significantly associated with an intention to attend a future dementia screening [adjusted OR (95% CI) = 0.51 (0.32-0.80)]. In both public and in-home screeners, high "perceived benefits" were significantly associated with an intention to attend a future dementia screening [adjusted OR (95% CI) = 2.13 (1.46-3.10); adjusted OR (95% CI) = 2.56 (1.22-5.35), respectively]. It is necessary to reduce "perceived severity" among in-home screeners to increase dementia screening participants.

Tooth loss-related dietary patterns and cognitive impairment in an elderly Japanese population: The Nakajima study.

Although several studies have demonstrated a potential correlation of dietary patterns with cognitive function, the relationship between tooth loss and dietary patterns and cognitive function have not been identified. In this cross-sectional study, we used a reduced rank regression (RRR) analysis, a technique used previously to observe dietary patterns based on the intakes of nutrients or levels of biomarkers associated with the condition of interest, to identify tooth loss-related dietary patterns and investigate the associations of such patterns with cognitive impairment in 334 community-dwelling Japanese subjects aged ≥ 60 years. According to Pearson correlation coefficients, the intakes of six nutrients (ash content, sodium, zinc, vitamin B1, α- and ß-carotene) correlated significantly with the number of remaining teeth. Using RRR analysis, we extracted four dietary patterns in our subject population that explained 86.67% of the total variation in the intakes of these six nutrients. Particularly, dietary pattern 1 (DP1) accounted for 52.2% of the total variation. Food groups with factor loadings of ≥ 0.2 included pickled green leafy vegetables, lettuce/cabbage, green leaves vegetables, cabbage, carrots/squash; by contrast, rice had a factor loading of <-0.2. In a multivariate regression analysis, the adjusted odds ratios regarding the prevalence of cognitive impairment for the lowest, middle and highest tertiles of the DP1 score were 1.00 (reference), 1.224 (95% confidence interval [CI]: 0.611-2.453) and 0.427 (95% CI: 0.191-0.954), respectively. To our knowledge, this is the first report to show that tooth loss-related dietary patterns are associated with a high prevalence of cognitive impairment. These results may motivate changes in dental treatment and the dietary behaviours and thereby lower the risk of cognitive impairment.

Auxin and ROP GTPase Signaling of Polar Nuclear Migration in Root Epidermal Hair Cells.

Polar nuclear migration is crucial during the development of diverse eukaryotes. In plants, root hair growth requires polar nuclear migration into the outgrowing hair. However, knowledge about the dynamics and the regulatory mechanisms underlying nuclear movements in root epidermal cells remains limited. Here, we show that both auxin and Rho-of-Plant (ROP) signaling modulate polar nuclear position at the inner epidermal plasma membrane domain oriented to the cortical cells during cell elongation as well as subsequent polar nuclear movement to the outer domain into the emerging hair bulge in Arabidopsis (Arabidopsis thaliana). Auxin signaling via the nuclear AUXIN RESPONSE FACTOR7 (ARF7)/ARF19 and INDOLE ACETIC ACID7 pathway ensures correct nuclear placement toward the inner membrane domain. Moreover, precise inner nuclear placement relies on SPIKE1 Rho-GEF, SUPERCENTIPEDE1 Rho-GDI, and ACTIN7 (ACT7) function and to a lesser extent on VTI11 vacuolar SNARE activity. Strikingly, the directionality and/or velocity of outer polar nuclear migration into the hair outgrowth along actin strands also are ACT7 dependent, auxin sensitive, and regulated by ROP signaling. Thus, our findings provide a founding framework revealing auxin and ROP signaling of inner polar nuclear position with some contribution by vacuolar morphology and of actin-dependent outer polar nuclear migration in root epidermal hair cells.

Serum anti-aquaporin-4 antibody-positive neuromyelitis optica spectrum disorder presenting as acute eosinophilic encephalomyelitis.

We report the case of a 57-year-old man with neuromyelitis optica spectrum disorder (NMOSD) presenting as acute eosinophilic encephalomyelitis. Magnetic resonance imaging revealed central nervous system lesions typical of NMOSD and anti-aquaporin-4 antibodies in the serum were identified; however, eosinophilia was evident in the cerebrospinal fluid (CSF) at the early stage of the disease. The number of eosinophils in the CSF decreased subsequently. Although activation of eosinophils is known to be an important factor in the development of NMOSD lesions, prominent eosinophilia in the CSF at the early stage of the disease has never been reported in patients with NMOSD.

Investigation of plasma induced electrical and chemical factors and their contribution processes to plasma gene transfection.

This study has been done to know what kind of factors in plasmas and processes on cells induce plasma gene transfection. We evaluated the contribution weight of three groups of the effects and processes, i.e. electrical, chemical and biochemical ones, inducing gene transfection. First, the laser produced plasma (LPP) was employed to estimate the contribution of the chemical factors. Second, liposomes were fabricated and employed to evaluate the effects of plasma irradiation on membrane under the condition without biochemical reaction. Third, the clathrin-dependent endocytosis, one of the biochemical processes was suppressed. It becomes clear that chemical factors (radicals and reactive oxygen/nitrogen species) do not work by itself alone and electrical factors (electrical current, charge and field) are essential to plasma gene transfection. It turned out the clathrin-dependent endocytosis is the process of the transfection against the 60% in all the transfected cells. The endocytosis and electrical poration are dominant in plasma gene transfection, and neither permeation through ion channels nor chemical poration is dominant processes. The simultaneous achievement of high transfection efficiency and high cell survivability is attributed to the optimization of the contribution weight among three groups of processes by controlling the weight of electrical and chemical factors.

Generation of CRISPR/Cas9-mediated bicistronic knock-in ins1-cre driver mice.

In the present study, we generated novel cre driver mice for gene manipulation in pancreatic ß cells. Using the CRISPR/Cas9 system, stop codon sequences of Ins1 were targeted for insertion of cre, including 2A sequences. A founder of C57BL/6J-Ins1(em1 (cre) Utr) strain was produced from an oocyte injected with pX330 containing the sequences encoding gRNA and Cas9 and a DNA donor plasmid carrying 2A-cre. (R26GRR x C57BL/6J-Ins1(em1 (cre) Utr)) F1 mice were histologically characterized for cre-loxP recombination in the embryonic and adult stages; cre-loxP recombination was observed in all pancreatic islets examined in which almost all insulin-positive cells showed tdsRed fluorescence, suggesting ß cell-specific recombination. Furthermore, there were no significant differences in results of glucose tolerance test among genotypes (homo/hetero/wild). Taken together, these observations indicated that C57BL/6J-Ins1(em1 (cre) Utr) is useful for studies of glucose metabolism and the strategy of bicistronic cre knock-in using the CRISPR/Cas9 system could be useful for production of cre driver mice.

Whole transcriptome analysis of transgenic barley with altered cytokinin homeostasis and increased tolerance to drought stress.

Cytokinin plant hormones have been shown to play an important role in plant response to abiotic stresses. Herein, we expand upon the findings of Pospísilová et al. [30] regarding preparation of novel transgenic barley lines overexpressing cytokinin dehydrogenase 1 gene from Arabidopsis under the control of mild root-specific promotor of maize ß-glycosidase. These lines showed drought-tolerant phenotype mainly due to alteration of root architecture and stronger lignification of root tissue. A detailed transcriptomic analysis of roots of transgenic plants subjected to revitalization after drought stress revealed attenuated response through the HvHK3 cytokinin receptor and up-regulation of two transcription factors implicated in stress responses and abscisic acid sensitivity. Increased expression of several genes involved in the phenylpropanoid pathway as well as of genes encoding arogenate dehydratase/lyase participating in phenylalanine synthesis was found in roots during revitalization. Although more precursors of lignin synthesis were present in roots after drought stress, final lignin accumulation did not change compared to that in plants grown under optimal conditions. Changes in transcriptome indicated a higher auxin turnover in transgenic roots. The same analysis in leaves revealed that genes encoding putative enzymes responsible for production of jasmonates and other volatile compounds were up-regulated. Although transgenic barley leaves showed lower chlorophyll content and down-regulation of genes encoding proteins involved in photosynthesis than did wild-type plants when cultivated under optimal conditions, they did show a tendency to return to initial photochemical activities faster than did wild-type leaves when re-watered after severe drought stress. In contrast to optimal conditions, comparative transcriptomic analysis of revitalized leaves displayed up-regulation of genes encoding enzymes and proteins involved in photosynthesis, and especially those encoded by the chloroplast genome. Taken together, our results indicate that the partial cytokinin insensitivity induced in barley overexpressing cytokinin dehydrogenase contributes to tolerance to drought stress.