Quantitative Analyses of Circadian Gene Expression in Mammalian Cell Cultures

The central circadian pacemaker is located in the hypothalamus of mammals, but essentially the same oscillating system operates in peripheral tissues and even in immortalized cell lines. Using luciferase reporters that allow automated monitoring of circadian gene expression in mammalian fibroblasts, we report the collection and analysis of precise rhythmic data from these cells. We use these methods to analyze signaling pathways of peripheral tissues by studying the responses of Rat-1 fibroblasts to ten different compounds. To quantify these rhythms, which show significant variation and large non-stationarities (damping and baseline drifting), we developed a new fast Fourier transform–nonlinear least squares analysis procedure that specifically optimizes the quantification of amplitude for circadian rhythm data. This enhanced analysis method successfully distinguishes among the ten signaling compounds for their rhythm-inducing properties. We pursued detailed analyses of the responses to two of these compounds that induced the highest amplitude rhythms in fibroblasts, forskolin (an activator of adenylyl cyclase), and dexamethasone (an agonist of glucocorticoid receptors). Our quantitative analyses clearly indicate that the synchronization mechanisms by the cAMP and glucocorticoid pathways are different, implying that actions of different genes stimulated by these pathways lead to distinctive programs of circadian synchronization

Impact of cognitive restructuring using narratives on automatic thoughts

研究1では，認知再構成法の要素を盛り込んだ物語を作成した。物語の題材は出雲（2016）より，友人関係，バイト関係，恋人関係で大学生に起こると思われる出来事をとりあげた。各物語には，認知再構成法の要素，「気分」「状況」「自動思考」「根拠」「反証」「適応的思考」「今の気分」を取り入れた。大学生，大学院生の計53名に物語の印象（気分，理解度，自己応用，わかりやすさ）の各項目について5件法で評定を行ってもらった。結果，友人関係，バイト関係の物語で全ての項目で3点以上だった。このことから，友人関係とバイト関係に関する物語が活用できることが示唆された。研究2では，研究1で作成した友人関係とバイト関係の物語を用いて，大学生と大学院生の9名に半構造化面接を行った。面接前にATQ-R 短縮版と日本語版PANAS に評定を行ってもらった。続いて，自分の悩みについての思考や気分について簡単に答えてもらい，認知再構成法の要素を盛り込んだ友人関係かバイト関係のいずれかの物語を読んでもらった。その後，再度，自分の悩みについての思考や気分について答え事後評定を行ってもらった。結果，否定的な思考や感情が有意に下がり，肯定的思考とポジティブ感情が上がることが示された。In study 1, narratives were created with the components of cognitive restructuring. The subjects for the narratives were events that are likely to arise among university students in friendships, part-time work relationships, and romantic relationships, as discussed by Izumo (2016). Each narrative included the components of cognitive restructuring: mood, status, automatic thoughts, evidence, counterevidence, adaptive thoughts, and current mood. A total of 53 university students and graduate students gave their impressions of the narratives by rating them on a five-point scale for each aspect (mood, comprehension, self-applicability, and comprehensibility). The friendship and part-time work relationship narratives scored three points or higher in all aspects. This indicated that the friendship and part-time work relationship narratives could be used.In study 2, the friendship and part-time work relationship narratives that were created in study 1 were used in semistructured interviews with nine university students and graduate students. Before the interviews, students completed the short-form ATQ-R and the Japanese version of the PANAS. They were then asked to give brief responses about their thoughts concerning their own problems and their mood and to read either the friendship narrative or the part-time work relationship narrative that both incorporated the components of cognitive restructuring. They were then asked again for their thoughts concerning their own problems and their mood and completed the assessments. The result was a significant decrease in negative thinking and emotions and an increase in positive thinking and emotions

Counseling scenes using the scene imagination method and the effects of different compliments on problem resolution

川口ら（2017）は，場面想定法を用いて大学生が学生相談室のカウンセラーから４つの異なるコンプリメントを行ってもらった場面を想定した条件での問題解決への取り組みを調べ，異なる影響が示唆された。本研究では，川口ら（2017）を参考に，大学生の自分の学業，友人関係，家族関係の各相談場面で，カウンセラーが内的リソースへの示唆，外的リソースへの示唆，努力への敬意，なしの各応答を行った場面を想定してもらい，インナーストレングス尺度とコンプリメントによる解決志向尺度に評定してもらった。結果，インナーストレングス尺度のつながり得点で相談内容とカウンセラー応答の交互作用が示された。友人関係では努力への敬意がなしより高く，自分の学業では外的リソースへの示唆が他の応答より高く，家族関係ではなしが他の応答より有意に低かった。このことから，友人関係では友達との関係を維持するための努力への敬意が，学業ではよい友人に恵まれていることへのコンプリメントによってつながり得点が高いことが示唆された。解決志向尺度の相手への信頼得点で交互作用が有意傾向で，友人関係で内的リソースへの示唆と努力への敬意がなしより高く，自分の学業と家族関係でなしが他の応答より低かった。このことから，コンプリメントがあることで信頼得点が高いことが示唆された。Kawaguchi et al. (2017) used the scene imagination method on university students to examine problem resolution actions in imaginary scenes of student counseling in which the counselor gave four types of compliments and found different effects. Based on that study, we gave university students imaginary counseling scenes concerning their school performance,friendships, and family relationships in which the counselor suggested they have internal resources, suggested external resources, respected their effort, or gave no compliment. Responses were evaluated using the Inner Strength Scale and the Solution-focused Inventory using compliments. The results showed an interaction between scenario and counselor response in the connectedness score on the Inner Strength Scale. For friendship, scores were higher when the counselor respected their effort compared to giving no compliment. For school performance, scores were highest when the counselor suggested they have external resources. For family relationships, scores were significantly lower when no compliments were received compared to when they received compliments. This suggests that respecting effort to maintain relationships with friends and complimenting great friendships in school performance increase connectedness score.Marginally significant difference of interaction was observed in scores for trusting others in the Solution-focused Inventory.In friendship, scores were higher when the counselor suggested they have internal resources or respected their effort compared to no compliment. In school performance and family relationships, scores were lower when receiving no compliment than when receiving a compliment of any kind. This suggests that trust score increases when a compliment is given

Effects of different finger-painting color on stress

下川床（2009）では，大学生を対象に12色の絵の具から自由に絵の具を選ぶ自由色群，黒色のみ選ぶ黒色群，統制群にランダムに分け，フィンガーペインティングを行ってもらった結果，自由色群においてフィンガーペインティングを行う前より行った後で，否定的感情や倦怠が有意に下がったことを示した。また，葛(2019) では，足し算を用いた単純作業を行う際の背景色の違いがストレスに及ぼす影響について検証した。結果，青色群と緑色群が赤色群よりもストレス状態の緊張覚醒得点が有意に低いことが示された。これらの研究結果を踏まえ，本研究では，2群（全色・青と緑）× 2テスト（事前・事後）の研究を計画した。大学生の参加者は，ランダムに全色群13名と青・緑群13名に分けられた。全色群は12色の絵の具から色を自由に使用してフィンガーペインティングを行った。青・緑色群は，青色と緑色の絵の具から自由に使用してフィンガーペインティングを行った。結果，エネルギー覚醒得点で青・緑色群の方が有意に低く，事前より事後で有意に低かった。なお，KH-Codre（樋口，2020）の分析により自由記述の共起ネットワーク図を作成した。結果，青・緑色群の共起ネットワークでは，「スッキリした」という感想が見られた。だが，ストレス尺度で交互作用が示されず，群の偏りがあったとも考えられ，今後，手続きについての課題も残る。In a study by Shimokawatoko (2009), university students were randomized into a free-choice color group where participants could freely choose from 12 lors of paint, a black group where only black paint could be used, and a control group, and were asked to paint with their fingers. Negative emotions and malaise significantly decreased in the free-choice color group after versus before finger painting. Ge (2019) investigated the effects of background color used in simple addition tasks on stress in which the blue and green group’s stress status had significantly lower tense arousal scores compared to the red group. Based on the aforementioned outcomes, we designed a study with two color options (all color and blue/green) × two tests (before and after). Participants were university students randomized into an all color group (n=13) and a blue/green group (n=13). The all color group could freely use 12 colors for finger painting while the blue/green group could freely use blue and green. The blue/green group had a significantly lower energy arousal score after versus before finger painting. In addition, co-occurrence network diagrams of text comments were created with KH-Coder (Higuchi, 2020) which showed that the co-occurrence network of the blue/green group included the comment, “It was refreshing.” However, no interaction on the stress scale was observed, indicating a potential group bias

Neuronal deletion of the circadian clock gene Bmal1 induces cell-autonomous dopaminergic neurodegeneration

Circadian rhythm dysfunction is a hallmark of Parkinson disease (PD), and diminished expression of the core clock gene Bmal1 has been described in patients with PD. BMAL1 is required for core circadian clock function but also serves nonrhythmic functions. Germline Bmal1 deletion can cause brain oxidative stress and synapse loss in mice, and it can exacerbate dopaminergic neurodegeneration in response to the toxin MPTP. Here we examined the effect of cell type-specific Bmal1 deletion on dopaminergic neuron viability in vivo. We observed that global, postnatal deletion of Bmal1 caused spontaneous loss of tyrosine hydroxylase+ (TH+) dopaminergic neurons in the substantia nigra pars compacta (SNpc). This was not replicated by light-induced disruption of behavioral circadian rhythms and was not induced by astrocyte- or microglia-specific Bmal1 deletion. However, either pan-neuronal or TH neuron-specific Bmal1 deletion caused cell-autonomous loss of TH+ neurons in the SNpc. Bmal1 deletion did not change the percentage of TH neuron loss after α-synuclein fibril injection, though Bmal1-KO mice had fewer TH neurons at baseline. Transcriptomics analysis revealed dysregulation of pathways involved in oxidative phosphorylation and Parkinson disease. These findings demonstrate a cell-autonomous role for BMAL1 in regulating dopaminergic neuronal survival and may have important implications for neuroprotection in PD

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

Schematic Representation of the Analysis Procedure

<p>One of the traces depicted in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020136#pcbi-0020136-g005" target="_blank">Figure 7</a> is shown. Raw data were first detrended by subtracting a 24-h moving average with uniform weight from raw data to produce zero-mean, zero-slope data. These subtracted data (DTR space) are further divided by the standard deviation to report the normalized detrended data in the SND space. These SND data then had a variance of 1, and were analyzed by FFT-NLLS to obtain period, phase, and RAE values. The figure shows an example of the fitted curve (green line, detrended raw data; red line, FFT-NLLS fitted curve). Based on these period and phase estimates, FFT-NLLS is performed on the subtracted data in the DTR space to solve for amplitude (α) between the fixed time range (36–60 h was used).</p

Fibroblast Rhythms Initiated by Various Treatments

<p>Confluent Rat1/P<i>mper2</i>::dLuc cells were treated for 2 h, then luminescence monitoring was begun. Time 0 is the onset of each treatment. DMSO (0.1%) is the solvent control for 1 μM ionomycin, 1 μM PMA, and 10 μM Fsk. Serum (0%) is a control for 50% horse serum. EtOH (0.001%) is the solvent control for 100 nM Dex. DMEM is the solvent control for 100 μM 8-bromo-cAMP, 30 nM EGF, 25 ng/ml bFGF, 30 nM ET-1, and 1 μM PGE₂.. Two representative traces from one experiment are shown in each graph.</p