Bioorthogonal chemical labeling of endogenous neurotransmitter receptors in living mouse brains

生きた動物脳内で発現する神経伝達物質受容体に目印を付ける新手法を開発 --遺伝子操作を伴わず、生体内でたんぱく質の機能解析が可能に--. 京都大学プレスリリース. 2024-02-05.Neurotransmitter receptors are essential components of synapses for communication between neurons in the brain. Because the spatiotemporal expression profiles and dynamics of neurotransmitter receptors involved in many functions are delicately governed in the brain, in vivo research tools with high spatiotemporal resolution for receptors in intact brains are highly desirable. Covalent labeling by chemical reaction (chemical labeling) of proteins without genetic manipulation is now a powerful method for analyzing receptors in vitro. However, selective target receptor labeling in the brain has not yet been achieved. This study shows that ligand-directed alkoxyacylimidazole (LDAI) chemistry can be used to selectively tether synthetic probes to target endogenous receptors in living mouse brains. The reactive LDAI reagents with negative charges were found to diffuse well over the whole brain and could selectively label target endogenous receptors, including AMPAR, NMDAR, mGlu1, and GABAAR. This simple and robust labeling protocol was then used for various applications: three-dimensional spatial mapping of endogenous receptors in the brains of healthy and disease-model mice; multi-color receptor imaging; and pulse–chase analysis of the receptor dynamics in postnatal mouse brains. Here, results demonstrated that bioorthogonal receptor modification in living animal brains may provide innovative molecular tools that contribute to the in-depth understanding of complicated brain functions

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)

Risk profiles and one-year outcomes of patients with newly diagnosed atrial fibrillation in India: Insights from the GARFIELD-AF Registry.

BACKGROUND: The Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF) is an ongoing prospective noninterventional registry, which is providing important information on the baseline characteristics, treatment patterns, and 1-year outcomes in patients with newly diagnosed non-valvular atrial fibrillation (NVAF). This report describes data from Indian patients recruited in this registry. METHODS AND RESULTS: A total of 52,014 patients with newly diagnosed AF were enrolled globally; of these, 1388 patients were recruited from 26 sites within India (2012-2016). In India, the mean age was 65.8 years at diagnosis of NVAF. Hypertension was the most prevalent risk factor for AF, present in 68.5% of patients from India and in 76.3% of patients globally (P < 0.001). Diabetes and coronary artery disease (CAD) were prevalent in 36.2% and 28.1% of patients as compared with global prevalence of 22.2% and 21.6%, respectively (P < 0.001 for both). Antiplatelet therapy was the most common antithrombotic treatment in India. With increasing stroke risk, however, patients were more likely to receive oral anticoagulant therapy [mainly vitamin K antagonist (VKA)], but average international normalized ratio (INR) was lower among Indian patients [median INR value 1.6 (interquartile range {IQR}: 1.3-2.3) versus 2.3 (IQR 1.8-2.8) (P < 0.001)]. Compared with other countries, patients from India had markedly higher rates of all-cause mortality [7.68 per 100 person-years (95% confidence interval 6.32-9.35) vs 4.34 (4.16-4.53), P < 0.0001], while rates of stroke/systemic embolism and major bleeding were lower after 1 year of follow-up. CONCLUSION: Compared to previously published registries from India, the GARFIELD-AF registry describes clinical profiles and outcomes in Indian patients with AF of a different etiology. The registry data show that compared to the rest of the world, Indian AF patients are younger in age and have more diabetes and CAD. Patients with a higher stroke risk are more likely to receive anticoagulation therapy with VKA but are underdosed compared with the global average in the GARFIELD-AF. CLINICAL TRIAL REGISTRATION-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01090362

Water Use and Growth of Maize under Water Stress on the Soil after Long-Term Applications of Chemical and/or Organic Fertilizers

Experiments were conducted in 1997 and 1999 to determine how long-term soil fertilization influences the performance of maize crop under water deficit on alluvial soil (sandy loam) at the Kyoto University Farm, Japan. The field had been applied large amounts of chemical fertilizer and/or farmyard manure since 1988 as four treatments, i. e., no fertilizer (NF), chemical fertilizer (CF), farmyard manure (MF) and chemical fertilizer + manure (CM) each with two replications. Water stress was imposed naturally in 1997 from 54 to 81 days after sowing (DAS). In 1999, water stress was imposed in one of the two replicate blocks artificially during 22-65 DAS by controlling irrigation under a rain shelter and another replicate block was well watered. Leaf area index and biomass in CM and MF were maintained higher than in CF and NF in both years under both water-stressed and well-watered conditions. With the progress of water stress, the major water depletion zone in the soil tended to shift deeper to a greater extent in MF and CM than in NF and CF. The total amount of water depleted from lOOcm-depth soil during the stress periods was larger in MF and CM than in NF and CF by 20 to 80 percent. Water-use efficiency (biomass production per total water depletion) was similar in CF, MF and CM with an average of 3.9 g kg–1 and it was lower in NF (1.8 g kg–1). Leaf conductance and photosynthetic rate during the stress period were higher in MF than in CF. The results indicated that water extraction during the stress period was greater in the plots manured for a long time, which resulted in well-maintained physiological activity and growth

Recovery of 15N-labeled Ammonium by Barley and Maize Grown on the Soils with Long-Term Application of Chemical and Organic Fertilizers

The fate of applied nitrogen (N) fertilizer in a barley-maize-barley crop sequence on an alluvial soil with for different histories of soil fertilization (main plots) was examined at Kyoto, Japan. The four main plots were not fertilized (NF) , or fertilized with only chemical fertilizer (CF) , only barnyard manure (MF) and chemical fertilizer + barnyard manure (GM) for nine years, and then two open-bottom boxes (mini-plot) were embedded in the middle of each main plot. 15N-labeled ammonium sulfate was applied to the mini-plots only before planting the first crop barley in group I, and before planting the first and the second crops in group 2. The highest biomass yield and Nuptake were observed in the GM main plot throughout the barley-maize-barley crop sequence, and in the MF main plot in the last two crops. In the mini-plots, recovery of 15N by the first barley ranged from 4%in MF to Il%in NF. After the third crop harvest, 11 to 23% of the labeled N remained in the soil in group I. The rate of remaining 15N was notably higher in MF and CM than in CF and NF. Labeled N applied before planting the second crop maize in group 2 also remained in the soil at a higher rate and recovered less by the plants in MF than in the other mini-plots.Recovery of applied N by the second crop maize in group 1, was less than 2%in all mini-plots. The contribution of the applied N to the total N uptake in the first crops was higher in NF and CF (24-48%) than in MF and CM (7-15%), and that to the subsequent crop was negligible in all mini-plots. In the soils previously fertilized with manure for nine years, the applied N was held in the soil more abundantly, but was less utilized by the plants grown in the above crop sequence than in the soil not fertilized with manure previously

Genotypic Variation in Ability to Recover from Weed Competition at Early Vegetative Stage in Upland Rice

In northern Laos, weeds are a major constraint to upland rice production in slash-and-burn systems. Two experiments were conducted to assess genotypic variation in ability to recover from weed competition at the early vegetative stage. Three traditional and two improved (IR 55423-01 and B6144F-MR-6-0-0) cultivars were grown with or without maize as an artificial weed. Maize was seeded at the same time as rice and removed at 37 d after rice sowing. The two improved cultivars out-yielded the traditional cultivars without weed competition. Larger yield loss due to the competition was associated with longer delay in days to flowering and smaller plant height at 37 d after sowing. The use of B6144F-MR-6-0-0 with high yield potential as well as strong ability to recover from weed competition appears to improve and stabilize rice productivity in this region

Ligand-directed two-step labeling to quantify neuronal glutamate receptor trafficking

The analysis of AMPA-type glutamate receptor (AMPAR) trafficking is essential for understanding molecular mechanisms of learning and memory, but the analytical tools are currently limited. Here, the authors report a method that combines affinity-based receptor labeling and bioorthogonal click chemistry to quantify AMPAR distribution and trafficking under physiological conditions