Low immunogenicity of LNP allows repeated administrations of CRISPR-Cas9 mRNA into skeletal muscle in mice

筋ジストロフィーのゲノム編集治療を目指したLNP-mRNA輸送システムの開発. 京都大学プレスリリース. 2021-12-08.Nanotechnology for genome editing in multiple muscles simultaneously. 京都大学プレスリリース. 2021-12-08.Genome editing therapy for Duchenne muscular dystrophy (DMD) holds great promise, however, one major obstacle is delivery of the CRISPR-Cas9/sgRNA system to skeletal muscle tissues. In general, AAV vectors are used for in vivo delivery, but AAV injections cannot be repeated because of neutralization antibodies. Here we report a chemically defined lipid nanoparticle (LNP) system which is able to deliver Cas9 mRNA and sgRNA into skeletal muscle by repeated intramuscular injections. Although the expressions of Cas9 protein and sgRNA were transient, our LNP system could induce stable genomic exon skipping and restore dystrophin protein in a DMD mouse model that harbors a humanized exon sequence. Furthermore, administration of our LNP via limb perfusion method enables to target multiple muscle groups. The repeated administration and low immunogenicity of our LNP system are promising features for a delivery vehicle of CRISPR-Cas9 to treat skeletal muscle disorders

Pipeline scheduling with input port constraints for an FPGA-based biochemical simulator

This paper discusses design methodology of high-throughput arithmetic pipeline modules for an FPGA-based biochemical simulator. Since limitation of data-input bandwidth caused by port constraints often has a negative impact on pipeline scheduling results, we propose a priority assignment method of input data which enables efficient arithmetic pipeline scheduling under given input port constraints. Evaluation results with frequently used rate-law functions in biochemical models revealed that the proposed method achieved shorter latency compared to ASAP and ALAP scheduling with random input orders, reducing hardware costs by 17.57% and by 27.43% on average, respectively.The original publication is available at www.springerlink.co

VEGF164-mediated Inflammation Is Required for Pathological, but Not Physiological, Ischemia-induced Retinal Neovascularization

Hypoxia-induced VEGF governs both physiological retinal vascular development and pathological retinal neovascularization. In the current paper, the mechanisms of physiological and pathological neovascularization are compared and contrasted. During pathological neovascularization, both the absolute and relative expression levels for VEGF(164) increased to a greater degree than during physiological neovascularization. Furthermore, extensive leukocyte adhesion was observed at the leading edge of pathological, but not physiological, neovascularization. When a VEGF(164)-specific neutralizing aptamer was administered, it potently suppressed the leukocyte adhesion and pathological neovascularization, whereas it had little or no effect on physiological neovascularization. In parallel experiments, genetically altered VEGF(164)-deficient (VEGF(120/188)) mice exhibited no difference in physiological neovascularization when compared with wild-type (VEGF(+/+)) controls. In contrast, administration of a VEGFk-1/Fc fusion protein, which blocks all VEGF isoforms, led to significant suppression of both pathological and physiological neovascularization. In addition, the targeted inactivation of monocyte lineage cells with clodronate-liposomes led to the suppression of pathological neovascularization. Conversely, the blockade of T lymphocyte-mediated immune responses with an anti-CD2 antibody exacerbated pathological neovascularization. These data highlight important molecular and cellular differences between physiological and pathological retinal neovascularization. During pathological neovascularization, VEGF(164) selectively induces inflammation and cellular immunity. These processes provide positive and negative angiogenic regulation, respectively. Together, new therapeutic approaches for selectively targeting pathological, but not physiological, retinal neovascularization are outlined

VEGF164-mediated Inflammation Is Required for Pathological, but Not Physiological, Ischemia-induced Retinal Neovascularization

Hypoxia-induced VEGF governs both physiological retinal vascular development and pathological retinal neovascularization. In the current paper, the mechanisms of physiological and pathological neovascularization are compared and contrasted. During pathological neovascularization, both the absolute and relative expression levels for VEGF164 increased to a greater degree than during physiological neovascularization. Furthermore, extensive leukocyte adhesion was observed at the leading edge of pathological, but not physiological, neovascularization. When a VEGF164-specific neutralizing aptamer was administered, it potently suppressed the leukocyte adhesion and pathological neovascularization, whereas it had little or no effect on physiological neovascularization. In parallel experiments, genetically altered VEGF164-deficient (VEGF120/188) mice exhibited no difference in physiological neovascularization when compared with wild-type (VEGF+/+) controls. In contrast, administration of a VEGFR-1/Fc fusion protein, which blocks all VEGF isoforms, led to significant suppression of both pathological and physiological neovascularization. In addition, the targeted inactivation of monocyte lineage cells with clodronate-liposomes led to the suppression of pathological neovascularization. Conversely, the blockade of T lymphocyte–mediated immune responses with an anti-CD2 antibody exacerbated pathological neovascularization. These data highlight important molecular and cellular differences between physiological and pathological retinal neovascularization. During pathological neovascularization, VEGF164 selectively induces inflammation and cellular immunity. These processes provide positive and negative angiogenic regulation, respectively. Together, new therapeutic approaches for selectively targeting pathological, but not physiological, retinal neovascularization are outlined

Why Are Outcomes Different for Registry Patients Enrolled Prospectively and Retrospectively? Insights from the Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF).

Background: Retrospective and prospective observational studies are designed to reflect real-world evidence on clinical practice, but can yield conflicting results. The GARFIELD-AF Registry includes both methods of enrolment and allows analysis of differences in patient characteristics and outcomes that may result. Methods and Results: Patients with atrial fibrillation (AF) and ≥1 risk factor for stroke at diagnosis of AF were recruited either retrospectively (n = 5069) or prospectively (n = 5501) from 19 countries and then followed prospectively. The retrospectively enrolled cohort comprised patients with established AF (for a least 6, and up to 24 months before enrolment), who were identified retrospectively (and baseline and partial follow-up data were collected from the emedical records) and then followed prospectively between 0-18 months (such that the total time of follow-up was 24 months; data collection Dec-2009 and Oct-2010). In the prospectively enrolled cohort, patients with newly diagnosed AF (≤6 weeks after diagnosis) were recruited between Mar-2010 and Oct-2011 and were followed for 24 months after enrolment. Differences between the cohorts were observed in clinical characteristics, including type of AF, stroke prevention strategies, and event rates. More patients in the retrospectively identified cohort received vitamin K antagonists (62.1% vs. 53.2%) and fewer received non-vitamin K oral anticoagulants (1.8% vs . 4.2%). All-cause mortality rates per 100 person-years during the prospective follow-up (starting the first study visit up to 1 year) were significantly lower in the retrospective than prospectively identified cohort (3.04 [95% CI 2.51 to 3.67] vs . 4.05 [95% CI 3.53 to 4.63]; p = 0.016). Conclusions: Interpretations of data from registries that aim to evaluate the characteristics and outcomes of patients with AF must take account of differences in registry design and the impact of recall bias and survivorship bias that is incurred with retrospective enrolment. Clinical Trial Registration: - URL: http://www.clinicaltrials.gov . Unique identifier for GARFIELD-AF (NCT01090362)

Effects of Observed Incubation Behavior on Egg Production in Laying Hens of a Commercial Chicken Breed and Detection of Single-Nucleotide Polymorphisms Associated with the Incubation Behavior

Upon contact with laid eggs, avians initiate incubation behavior and stop laying additional eggs. This phenomenon suggests that the productivity of laying hens in free-range facilities may decrease because of frequent contact with laid eggs. Here, we examined whether hens of a commercial breed exhibit incubation behavior in a free-range facility and whether egg productivity subsequently decreases. One-hour observations were performed twice weekly for 3 weeks, during which 9 of 129 hens (7.0%) exhibited incubation behavior (i.e., sitting on eggs) in the free-range facility and were defined as incubating hens. During 4 d of continuous behavioral observation, incubating and non-incubating hens laid the same number of eggs statistically (4.6 and 3.6, on average, respectively); however, incubating hens spent significantly more time on average incubating the eggs (2071.9 min) than did the non-incubating hens (20.9 min; P<0.05), indicating a clear behavioral difference. Subsequently, the incubation behavior and egg productivity of incubating hens and a Silkie Fowl breed hen, which is known to exhibit typical incubation behavior and cessation of laying, were continuously compared for 27 d. The average minutes spent incubating eggs during the observation period increased in both the incubating hens and Silkie Fowl hen and the total time was almost the same (18,088.5 and 23,092 min, respectively). However, the Silkie Fowl hen stopped laying on day 17 after laying 17 eggs, whereas the incubating hens continued laying throughout the observation period. Incubating hens laid an average of 24.5 eggs, indicating that some hens (at least those of the commercial breed used in our study) can continue laying while exhibiting incubation behavior. A single-nucleotide polymorphism associated with incubation behavior was detected on chromosome 4 through genome-wide association analysis

Effects of Observed Incubation Behavior on Egg Production in Laying Hens of a Commercial Chicken Breed and Detection of Single-Nucleotide Polymorphisms Associated with the Incubation Behavior

Upon contact with laid eggs, avians initiate incubation behavior and stop laying additional eggs. This phenomenon suggests that the productivity of laying hens in free-range facilities may decrease because of frequent contact with laid eggs. Here, we examined whether hens of a commercial breed exhibit incubation behavior in a free-range facility and whether egg productivity subsequently decreases. One-hour observations were performed twice weekly for 3 weeks, during which 9 of 129 hens (7.0%) exhibited incubation behavior (i.e., sitting on eggs) in the free-range facility and were defined as incubating hens. During 4 d of continuous behavioral observation, incubating and non-incubating hens laid the same number of eggs statistically (4.6 and 3.6, on average, respectively); however, incubating hens spent significantly more time on average incubating the eggs (2071.9 min) than did the non-incubating hens (20.9 min; P&lt;0.05), indicating a clear behavioral difference. Subsequently, the incubation behavior and egg productivity of incubating hens and a Silkie Fowl breed hen, which is known to exhibit typical incubation behavior and cessation of laying, were continuously compared for 27 d. The average minutes spent incubating eggs during the observation period increased in both the incubating hens and Silkie Fowl hen and the total time was almost the same (18,088.5 and 23,092 min, respectively). However, the Silkie Fowl hen stopped laying on day 17 after laying 17 eggs, whereas the incubating hens continued laying throughout the observation period. Incubating hens laid an average of 24.5 eggs, indicating that some hens (at least those of the commercial breed used in our study) can continue laying while exhibiting incubation behavior. A single-nucleotide polymorphism associated with incubation behavior was detected on chromosome 4 through genome-wide association analysis

Nicotinamide mononucleotide induces lipolysis by regulating ATGL expression via the SIRT1-AMPK axis in adipocytes

Nicotinamide adenine dinucleotide (NAD+) -dependent protein deacetylase SIRT1 plays an important role in the regulation of metabolism. Although the administration of nicotinamide mononucleotide (NMN), a key NAD+ intermediate, has been shown to ameliorate metabolic disorders, such as insulin resistance and glucose intolerance, the direct effect of NMN on the regulation of lipid metabolism in adipocytes remains unclear. We here investigated the effect of NMN on lipid storage in 3T3-L1 differentiated adipocytes. Oil-red O staining showed that NMN treatment reduced lipid accumulation in these cells. NMN was found to enhance lipolysis in adipocytes since the concentration of glycerol in the media was increased by NMN treatment. Western blotting and real-time RT-PCR analysis revealed that adipose triglyceride lipase (ATGL) expression at both protein and mRNA level was increased with NMN treatment in 3T3-L1 adipocytes. Whereas NMN increased SIRT1 expression and AMPK activation, an AMPK inhibitor compound C restored the NMN-dependent upregulation of ATGL expression in these cells, suggesting that NMN upregulates ATGL expression through the SIRT1-AMPK axis. NMN administration significantly decreased subcutaneous fat mass in mice on a high-fat diet. We also found that adipocyte size in subcutaneous fat was decreased with NMN treatment. Consistent with the alteration of fat mass and adipocyte size, the ATGL expression in subcutaneous fat was slightly, albeit significantly, increased with NMN treatment. These results indicate that NMN suppresses subcutaneous fat mass in diet-induced obese mice, potentially in part via the upregulation of ATGL. Unexpectedly, the reduction in fat mass as well as ATGL upregulation with NMN treatment were not observed in epididymal fat, implying that the effects of NMN are site-specific in adipose tissue. Thus, these findings provide important insights into the mechanism of NMN/NAD+ in the regulation of metabolism