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Yukiko Shiro,1,2 Young-Chang Arai,1 Yuichiro Nakaso,1 Hiroki Sakurai,1,3 Masayuki Inoue,1 Keiko Owari,1 Jun Sato,1 Tatsunori Ikemoto,4 Takahiro Ushida1 1Department of Pain Medicine, Aichi Medical University, Aichi, Japan; 2Department of Physical Therapy, Faculty of Rehabilitation Sciences, Nagoya Gakuin University, Aichi, Japan; 3Faculty of Health and Medical Sciences, Tokoha University, Shizuoka, Japan; 4Department of Orthopedics, School of Medicine, Aichi Medical University, Aichi, JapanCorrespondence: Yukiko Shiro, Nagoya Gakuin University, 3-1-17 Taiho Atsuta, Nagoya, Aichi, 456-0062, Japan, Tel +81 52-676-4078, Email siro823@ngu.ac.jpBackground: There are many factors associated with chronic pain, including changes in the nervous and musculoskeletal systems and so on. Recently, it has become clear that the gut microbiota (GM) influences these factors, and there are many reports of GM dysbiosis in patients with chronic pain. However, the relationship between pain and GM remains unclear. Our previous study reported that defecation status, which reflects GM composition, was associated with pain intensity and that this relationship was different for each pain site. Our study investigated the association between pain site and the GM composition of feces in chronic pain patients.Methods: The subjects were 136 patients with chronic pain and 125 healthy controls. Patients were classified into four groups, whole body (WB) pain, lower back and lower extremity (LL) pain, headache, and upper back and upper extremity pain, based on the site of pain, and we investigated differences in GM taxonomy groups compared with healthy subject.Results: Chronic pain patients had a lower alpha diversity (effect size=0.16, p=0.02). But each pain site group did not differ in alpha diversity. WB pain patients showed higher Eggerthellaceae (LDA=3.09, p< 0.01) and lower Halomonas (LDA =− 2.72, p< 0.01). LL pain patients had increased Fusobacterium and Sellimonas (LDA=4.09,3.03 p< 0.01, 0.01) but reduced Halomonas (LDA=− 2.59, p< 0.01), and other key taxa.Conclusion: WB and LL patients may have GM compositions different from healthy controls, but larger studies are needed to confirm this.Keywords: gut microbiota, chronic pain, whole body pain, low back pain

Yukiko Shiro,1 Tatsunori Ikemoto,2 Kazuhiro Hayashi,3 Young-Chang Arai,4 Masataka Deie,2 Takefumi Ueno5 1Department of Physical Therapy, Faculty of Rehabilitation Sciences, Nagoya Gakuin University, Aichi, Japan; 2Department of Orthopaedics, Aichi Medical University, Aichi, Japan; 3Department of Rehabilitation, Aichi Medical University, Aichi, Japan; 4Institute of Physical Fitness, Sports Medicine and Rehabilitation, School of Medicine, Aichi Medical University, Aichi, Japan; 5National Hospital Organization Hizen Psychiatric Center, Saga, Japan Aim: Operant conditioning has long been believed to influence the pain experience through a psychological reward pathway. This study was formulated to test the hypothesis that pain sensitivity may be enhanced >3 months if a monetary reward works as a reinforcement.Methods: Forty healthy subjects volunteered to participate in this study. The subjects repeatedly underwent pain testing via mechanical stimuli, and they rolled dice three (or six) times to gain money at the following five time points: baseline, three reinforcement sessions, and last session. The payoff was determined by roll of the dice. The subjects were instructed to roll the dice into a masked stand three times per session and informed that no one monitored the number of dice actually appeared. The subjects were also informed that they could roll the dice another three times when they reported strong pain during reinforcement sessions.Results: The amount of individual payoff had significantly increased at last session compared with the values obtained at baseline; however, no changes were identified in terms of the pain ratings for mechanical stimuli during all sessions.Conclusion: The results suggest that the psychological reward pathway does not always involve pain perception, and it is difficult to conclude whether pain sensitivity is operantly changed through the monetary reward in healthy individuals. Further investigation is required. Keywords: pain sensitivity, chronic pain, behavioral manipulation, reinforcement

Abstract Background Stage 4 neuroblastoma (NBL), a solid tumor of childhood, has a poor prognosis. Despite intensive molecular genetic studies, no targetable gene abnormalities have been identified. Stage 4S NBL has a characteristic of spontaneous regression, and elucidation of the mechanistic differences between stages 4 and 4S may improve treatment. Conventional NBL studies have mainly focused on the detection of abnormalities in individual genes and have rarely examined abnormalities in gene networks. While the gene coexpression network is expected to contribute to the detection of network abnormalities, the fragility of the network due to data noise and the extraction of arbitrary topological structures for the high-dimensional network are issues. Results The present paper concerns the classification method of stages 4 and 4S NBL patients using highly accurate gene coexpression network analysis based on RNA-sequencing data of transcription factors (TFs). In particular, after applying a noise reduction method RECODE, generalized topological overlapping measure (GTOM), which weighs the connections of nodes in the network structure, succeeded in extracting a cluster of TFs that showed high classification performance for stages 4 and 4S. In addition, we investigated how these clusters correspond to clinical information and to TFs which control the normal adrenal tissue and NBL characters. Conclusions A clustering method is presented for finding intermediate-scale clusters of TFs that give considerable separation performance for distinguishing between stages 4 and 4S. It is suggested that this method is useful as a way to extract factors that contribute to the separation of groups from multiple pieces of information such as gene expression levels.

BackgroundThe addition of antiplatelet therapy to anticoagulant therapy in patients with stroke with non-valvular atrial fibrillation (NVAF) and atherothrombotic disease may increase bleeding risk without reducing recurrent stroke risk.AimsTo evaluate the clinical benefits of anticoagulant monotherapy compared to combination therapy with anticoagulants and antiplatelet agents.Methods and designThis is an investigator-initiated prospective multicenter, randomized, open-label, parallel-group clinical trial. Patients with NVAF and atherothrombotic disease who have had a recent ischemic stroke or transient ischemic attack will be eligible to participate in this trial.Study outcomesThe primary outcome is a composite of ischemic cardiovascular events, including cardiovascular death, ischemic stroke, myocardial infarction, systemic embolism, ischemic events requiring urgent revascularization, and major bleeding events within 2 years after randomization.Sample size estimatesThis study will enroll 400 patients, 200 receiving anticoagulant monotherapy and 200 receiving combination therapy. This sample size will provide 90% power (one-sided p = 0.025) to detect a risk reduction in outcome events within 2 years, assuming event rates of 13 and 27% for each group, respectively, and a 10% loss to follow-up at a 2.5% significance level with one-sided log-rank tests at an interim analysis and a final analysis.DiscussionThis will be the first study to assess the net clinical benefit of oral anticoagulant monotherapy in ischemic stroke patients with NVAF and atherothrombosis.Clinical trial registrationhttps://clinicaltrials.gov/study/NCT03062319, NCT03062319; https://center6.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000029222, UMIN000025392; https://jrct.niph.go.jp/latest-detail/jRCTs051180202, jRCTs051180202.

High B-type natriuretic peptide (BNP) levels are associated with new atrial fibrillation (AF). This study investigated the distribution of AF detection rates according to BNP levels in patients with cryptogenic stroke (CS) using an insertable cardiac monitor (ICM). We enrolled consecutive patients with CS who underwent ICM implantation between October 2016 and September 2020 at eight stroke centers in Japan. Those with BNP levels were divided into three groups by tertiles. We evaluated the association of BNP levels with AF detection. Youden’s index was calculated to identify the optimal cutoff for BNP. Of 417 patients, we analyzed 266 patients with BNP data. The tertile range of BNP level was 19.0 to 48.5 pg/mL. AF detection rate was 13.3%/year, 12.8%/year, and 53.7%/year in the low-BNP (≤19.0), mid-BNP (19.1–48.4), and high-BNP (≥48.5) groups, respectively (log-rank trend p

Summary: In normal intestines, a fetal/regenerative/revival cell state can be induced upon inflammation. This plasticity in cell fate is also one of the current topics in human colorectal cancer (CRC). To dissect the underlying mechanisms, we generated human CRC organoids with naturally selected genetic mutation profiles and exposed them to two different conditions by modulating the extracellular matrix (ECM). Among tested mutation profiles, a fetal/regenerative/revival state was induced following YAP activation via a collagen type I-enriched microenvironment. Mechanistically, YAP transcription was promoted by activating AP-1 and TEAD-dependent transcription and suppressing intestinal lineage-determining transcription via mechanotransduction. The phenotypic conversion was also involved in chemoresistance, which could be potentially resolved by targeting the underlying YAP regulatory elements, a potential target of CRC treatment.

Abstract Background The emerging concepts of fetal-like reprogramming following tissue injury have been well recognized as an important cue for resolving regenerative mechanisms of intestinal epithelium during inflammation. We previously revealed that the remodeling of mesenchyme with collagen fibril induces YAP/TAZ-dependent fate conversion of intestinal/colonic epithelial cells covering the wound bed towards fetal-like progenitors. To fully elucidate the mechanisms underlying the link between extracellular matrix (ECM) remodeling of mesenchyme and fetal-like reprogramming of epithelial cells, it is critical to understand how collagen type I influence the phenotype of epithelial cells. In this study, we utilize collagen sphere, which is the epithelial organoids cultured in purified collagen type I, to understand the mechanisms of the inflammatory associated reprogramming. Resolving the entire landscape of regulatory networks of the collagen sphere is useful to dissect the reprogrammed signature of the intestinal epithelium. Methods We performed microarray, RNA-seq, and ATAC-seq analyses of the murine collagen sphere in comparison with Matrigel organoid and fetal enterosphere (FEnS). We subsequently cultured human colon epithelium in collagen type I and performed RNA-seq analysis. The enriched genes were validated by gene expression comparison between published gene sets and immunofluorescence in pathological specimens of ulcerative colitis (UC). Results The murine collagen sphere was confirmed to have inflammatory and regenerative signatures from RNA-seq analysis. ATAC-seq analysis confirmed that the YAP/TAZ-TEAD axis plays a central role in the induction of the distinctive signature. Among them, TAZ has implied its relevant role in the process of reprogramming and the ATAC-based motif analysis demonstrated not only Tead proteins, but also Fra1 and Runx2, which are highly enriched in the collagen sphere. Additionally, the human collagen sphere also showed a highly significant enrichment of both inflammatory and fetal-like signatures. Immunofluorescence staining confirmed that the representative genes in the human collagen sphere were highly expressed in the inflammatory region of ulcerative colitis. Conclusions Collagen type I showed a significant influence in the acquisition of the reprogrammed inflammatory signature in both mice and humans. Dissection of the cell fate conversion and its mechanisms shown in this study can enhance our understanding of how the epithelial signature of inflammation is influenced by the ECM niche.

NANOS3 is an evolutionarily conserved gene expressed in primordial germ cells that is important for germ cell development. Germ cell deletion by NANOS3 knockout has been reported in several mammalian species, but its function in pigs is unclear. In the present study, we investigated the germline effects of NANOS3 knockout in pigs using CRISPR/Cas9. Embryo transfer of CRISPR/Cas9-modified embryos produced ten offspring, of which one showed wild-type NANOS3 alleles, eight had two mutant NANOS3 alleles, and the other exhibited mosaicism (four mutant alleles). Histological analysis revealed no germ cells in the testes or ovaries of any of the nine mutant pigs. These results demonstrated that NANOS3 is crucial for porcine germ cell production.