Time-resolved studies of metalloproteins using X-ray free electron laser radiation at SACLA

Background: The invention of the X-ray free-electron laser (XFEL) has provided unprecedented new opportunities for structural biology. The advantage of XFEL is an intense pulse of X-rays and a very short pulse duration ( Scope of review: Recent time-resolved crystallographic analyses in XFEL facility SACLA are reviewed. Specifically, metalloproteins involved in the essential reactions of bioenergy conversion including photosystem II, cytochrome c oxidase and nitric oxide reductase are described. Major conclusions: XFEL with pump-probe techniques successfully visualized the process of the reaction and the dynamics of a protein. Since the active center of metalloproteins is very sensitive to the X-ray radiation, damage-free structures obtained by XFEL are essential to draw mechanistic conclusions. Methods and tools for sample delivery and reaction initiation are key for successful measurement of the time-resolved data. General significance: XFEL is at the center of approaches to gain insight into complex mechanism of structural dynamics and the reactions catalyzed by biological macromolecules. Further development has been carried out to expand the application of time-resolved X-ray crystallography. This article is part of a Special Issue entitled Novel measurement techniques for visualizing 'live' protein molecules

Diet-induced obesity in zebrafish shares common pathophysiological pathways with mammalian obesity

<p>Abstract</p> <p>Background</p> <p>Obesity is a multifactorial disorder influenced by genetic and environmental factors. Animal models of obesity are required to help us understand the signaling pathways underlying this condition. Zebrafish possess many structural and functional similarities with humans and have been used to model various human diseases, including a genetic model of obesity. The purpose of this study was to establish a zebrafish model of diet-induced obesity (DIO).</p> <p>Results</p> <p>Zebrafish were assigned into two dietary groups. One group of zebrafish was overfed with <it>Artemia </it>(60 mg dry weight/day/fish), a living prey consisting of a relatively high amount of fat. The other group of zebrafish was fed with <it>Artemia </it>sufficient to meet their energy requirements (5 mg dry weight/day/fish). Zebrafish were fed under these dietary protocols for 8 weeks. The zebrafish overfed with <it>Artemia </it>exhibited increased body mass index, which was calculated by dividing the body weight by the square of the body length, hypertriglyceridemia and hepatosteatosis, unlike the control zebrafish. Calorie restriction for 2 weeks was applied to zebrafish after the 8-week overfeeding period. The increased body weight and plasma triglyceride level were improved by calorie restriction. We also performed comparative transcriptome analysis of visceral adipose tissue from DIO zebrafish, DIO rats, DIO mice and obese humans. This analysis revealed that obese zebrafish and mammals share common pathophysiological pathways related to the coagulation cascade and lipid metabolism. Furthermore, several regulators were identified in zebrafish and mammals, including APOH, IL-6 and IL-1β in the coagulation cascade, and SREBF1, PPARα/γ, NR1H3 and LEP in lipid metabolism.</p> <p>Conclusion</p> <p>We established a zebrafish model of DIO that shared common pathophysiological pathways with mammalian obesity. The DIO zebrafish can be used to identify putative pharmacological targets and to test novel drugs for the treatment of human obesity.</p

Effects of multicomponent exercise on cognitive function in older adults with amnestic mild cognitive impairment: a randomized controlled trial

BACKGROUND: To examine the effects of a multicomponent exercise program on the cognitive function of older adults with amnestic mild cognitive impairment (aMCI). METHODS: Design: Twelve months, randomized controlled trial; Setting: Community center in Japan; Participants: Fifty older adults (27 men) with aMCI ranging in age from 65 to 93 years (mean age, 75 years); Intervention: Subjects were randomized into either a multicomponent exercise (n = 25) or an education control group (n = 25). Subjects in the multicomponent exercise group exercised under the supervision of physiotherapists for 90 min/d, 2 d/wk, for a total of 80 times over 12 months. The exercises included aerobic exercises, muscle strength training, and postural balance retraining, and were conducted using multiple conditions to stimulate cognitive functions. Subjects in the control group attended three education classes regarding health during the 12-month period. Measurements were administered before, after the 6-month, and after the 12-month intervention period; Measurements: The performance measures included the mini-mental state examination, logical memory subtest of the Wechsler memory scale-revised, digit symbol coding test, letter and categorical verbal fluency test, and the Stroop color word test. RESULTS: The mean adherence to the exercise program was 79.2%. Improvements of cognitive function following multicomponent exercise were superior at treatment end (group × time interactions for the mini-mental state examination (P = 0.04), logical memory of immediate recall (P = 0.03), and letter verbal fluency test (P = 0.02)). The logical memory of delayed recall, digit symbol coding, and Stroop color word test showed main effects of time, although there were no group × time interactions. CONCLUSIONS: This study indicates that exercise improves or supports, at least partly, cognitive performance in older adults with aMCI

A Large, Cross-Sectional Observational Study of Serum BDNF, Cognitive Function, and Mild Cognitive Impairment in the Elderly

Objective: The clinical relationship between brain-derived neurotrophic factor (BDNF) and cognitive function or mild cognitive impairment (MCI) is not well understood. The purpose of this study was to identify the relationship between serum BDNF and cognitive function and MCI, and determine whether serum BDNF level might be a useful biomarker for assessing risk for MCI in older people.Materials and Methods: A total of 4463 individuals aged 65 years or older (mean age 72 years) participating in the study. We measured performance in a battery of neuropsychological and cognitive function tests; serum BDNF concentration.Results: Eight hundred twenty-seven participants (18.8%) had MCI. After adjustment for sex, age, education level, diabetes, and current smoking, serum BDNF was associated with poorer performance in the story memory, and digit symbol substitution task scores. Serum BDNF was marginally associated with the presence of MCI (OR, 95% CI: 1.41, 1.00–1.99) when BDNF was 1.5 SD lower than the mean value standardized for sex and age, education level, diabetes, and current smoking.Conclusion: Low serum BDNF was associated with lower cognitive test scores and MCI. Future prospective studies should establish the discriminative value of serum BDNF for the risk of MCI

Terpene down-regulation triggers defense responses in transgenic oranges leading to resistance against fungal pathogens

Terpenoid volatiles are isoprene compounds that are emitted by plants to communicate with the environment. In addition to their function in repelling herbivores and attracting carnivorous predators in green tissues, the presumed primary function of terpenoid volatiles released from mature fruits is the attraction of seed-dispersing animals. Mature oranges (Citrus sinensis) primarily accumulate terpenes in peel oil glands, with D-limonene accounting for approximately 97% of the total volatile terpenes. In a previous report, we showed that down-regulation of a D-limonene synthase gene alters monoterpene levels in orange antisense (AS) fruits, leading to resistance against Penicillium digitatum infection. A global gene expression analysis of AS versus empty vector (EV) transgenic fruits revealed that the down-regulation of D-limonene up-regulated genes involved in the innate immune response. Basal levels of jasmonic acid were substantially higher in the EV compared with AS oranges. Upon fungal challenge, salicylic acid levels were triggered in EV samples, while jasmonic acid metabolism and signaling were drastically increased in AS orange peels. In nature, D-limonene levels increase in orange fruit once the seeds are fully viable. The inverse correlation between the increase in D-limonene content and the decrease in the defense response suggests that D-limonene promotes infection by microorganisms that are likely involved in facilitating access to the pulp for seed-dispersing frugivores.This work was supported by the Fondo Europeo de Desarrollo Regional and the Ministry of Science and Innovation of Spain (grant no. AGL2009-08052), by the Fundo de Defesa da Citricultura, and by the Instituto Valenciano de Investigaciones Agrarias, Spain (Ph.D. fellowship to A.R.).Rodríguez, A.; Shimada, T.; Cervera, M.; Alquézar, B.; Gadea Vacas, J.; Gómez-Cadenas, A.; De Ollas, CJ.... (2014). Terpene down-regulation triggers defense responses in transgenic oranges leading to resistance against fungal pathogens. Plant Physiology. 164(1):321-339. https://doi.org/10.1104/pp.113.224279S321339164

In vivo imaging of zebrafish retinal cells using fluorescent coumarin derivatives

<p>Abstract</p> <p>Background</p> <p>The zebrafish visual system is a good research model because the zebrafish retina is very similar to that of humans in terms of the morphologies and functions. Studies of the retina have been facilitated by improvements in imaging techniques. <it>In vitro </it>techniques such as immunohistochemistry and <it>in vivo </it>imaging using transgenic zebrafish have been proven useful for visualizing specific subtypes of retinal cells. In contrast, <it>in vivo </it>imaging using organic fluorescent molecules such as fluorescent sphingolipids allows non-invasive staining and visualization of retinal cells <it>en masse</it>. However, these fluorescent molecules also localize to the interstitial fluid and stain whole larvae.</p> <p>Results</p> <p>We screened fluorescent coumarin derivatives that might preferentially stain neuronal cells including retinal cells. We identified four coumarin derivatives that could be used for <it>in vivo </it>imaging of zebrafish retinal cells. The retinas of living zebrafish could be stained by simply immersing larvae in water containing 1 μg/ml of a coumarin derivative for 30 min. By using confocal laser scanning microscopy, the lamination of the zebrafish retina was clearly visualized. Using these coumarin derivatives, we were able to assess the development of the zebrafish retina and the morphological abnormalities induced by genetic or chemical interventions. The coumarin derivatives were also suitable for counter-staining of transgenic zebrafish expressing fluorescent proteins in specific subtypes of retinal cells.</p> <p>Conclusions</p> <p>The coumarin derivatives identified in this study can stain zebrafish retinal cells in a relatively short time and at low concentrations, making them suitable for <it>in vivo </it>imaging of the zebrafish retina. Therefore, they will be useful tools in genetic and chemical screenings using zebrafish to identify genes and chemicals that may have crucial functions in the retina.</p

Impact of D-limonene synthase up- or down-regulation on sweet orange fruit and juice odor perception

[EN] Citrus fruits are characterized by a complex mixture of volatiles making up their characteristic aromas, being the D-limonene the most abundant one. However, its role on citrus fruit and juice odor is controversial. Transgenic oranges engineered for alterations in the presence or concentration of few related chemical groups enable asking precise questions about their contribution to overall odor, either positive or negative, as perceived by the human nose. Here, either down- or up-regulation of a D-limonene synthase allowed us to infer that a decrease of as much as 51 times in D-limonene and an increase of as much as 3.2 times in linalool in juice were neutral for odor perception while an increase of only 3 times in ethyl esters stimulated the preference of 66% of the judges. The ability to address these questions presents exciting opportunities to understand the basic principles of selection of food. (C) 2016 Elsevier Ltd. All rights reserved.We are grateful to Drs. Lorenzo Zacarias and M. Jesus Rodrigo (IATA-CSIC) for the GC-MS facilities and support. We would like to acknowledge also to Drs. Berta Alquezar and Elsa Pons for their critical review of the manuscript and to all the panelists that participated in the sensory panel. This research is being funded in part by Fundo de Defesa da Citricultura (Fundecitrus).Rodríguez-Baixauli, AM.; Peris-Rodrigo, JE.; Redondo, A.; Shimada, T.; Costell, E.; Carbonell, I.; Rojas, C.... (2017). Impact of D-limonene synthase up- or down-regulation on sweet orange fruit and juice odor perception. Food Chemistry. 217:139-150. doi:10.1016/j.foodchem.2016.08.076S13915021

The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force

「コロナ制圧タスクフォース」COVID-19患者由来の血液細胞における遺伝子発現の網羅的解析 --重症度に応じた遺伝子発現の変化には、ヒトゲノム配列の個人差が影響する--. 京都大学プレスリリース. 2022-08-23.Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target