Serine metabolism is crucial for cGAS-STING signaling and viral defense control in the gut.

Inflammatory bowel diseases are characterized by the chronic relapsing inflammation of the gastrointestinal tract. While the molecular causality between endoplasmic reticulum (ER) stress and intestinal inflammation is widely accepted, the metabolic consequences of chronic ER stress on the pathophysiology of IBD remain unclear. By using in vitro, in vivo models, and patient datasets, we identified a distinct polarization of the mitochondrial one-carbon metabolism and a fine-tuning of the amino acid uptake in intestinal epithelial cells tailored to support GSH and NADPH metabolism upon ER stress. This metabolic phenotype strongly correlates with IBD severity and therapy response. Mechanistically, we uncover that both chronic ER stress and serine limitation disrupt cGAS-STING signaling, impairing the epithelial response against viral and bacterial infection and fueling experimental enteritis. Consequently, the antioxidant treatment restores STING function and virus control. Collectively, our data highlight the importance of serine metabolism to allow proper cGAS-STING signaling and innate immune responses upon gut inflammation.

High-intensity interval training improves respiratory and cardiovascular adjustments before and after initiation of exercise.

Purpose: High-intensity interval training (HIIT) may induce training-specific physiological adaptations such as improved respiratory and cardiovascular adjustments before and after the onset of high-intensity exercise, leading to improved exercise performance during high-intensity exercise. The present study investigated the effects of HIIT on time-dependent cardiorespiratory adjustment during maximal exercise and before and after initiation of high-intensity exercise, as well as on maximal exercise performance. Methods: 21 healthy male college students were randomly assigned to HIIT group (n = 11) or control group (n = 10). HIIT group performed training on a cycle ergometer once a week for 8 weeks. The training consisted of three bouts of exercise at 95% maximal work rate (WRmax) until exhaustion. Before and after the HIIT program, dynamic cardiorespiratory function was investigated by ramp and step exercise tests, and HIIT-induced cardiac morphological changes were assessed using echocardiography. Results: HIIT significantly improved not only maximal oxygen uptake and minute ventilation, but also maximal heart rate (HR), systolic blood pressure (SBP), and time to exhaustion in both exercise tests (p < 0.05). Time-dependent increases in minute ventilation (VE) and HR before and at the start of exercise were significantly enhanced after HIIT. During high-intensity exercise, there was a strong correlation between percent change (from before to after HIIT program) in time to exhaustion and percent change in HRmax (r = 0.932, p < 0.001). Furthermore, HIIT-induced cardiac morphological changes such as ventricular wall hypertrophy was observed (p < 0.001). Conclusion: We have demonstrated that HIIT at 95% WRmax induces training-specific adaptations such as improved cardiorespiratory adjustments, not only during maximal exercise but also before and after the onset of high-intensity exercise, improvement of exercise performance mainly associated with circulatory systems.

Discovery of non-genomic drivers of YAP signaling modulating the cell plasticity in CRC tumor lines.

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.

Single cell analysis revealed that two distinct, unique CD4+ T cell subsets were increased in the small intestinal intraepithelial lymphocytes of aged mice.

Recent advances in research suggest that aging has a controllable chronic inflammatory disease aspect. Aging systemic T cells, which secrete pro-inflammatory factors, affect surrounding somatic cells, and accelerate the aging process through chronic inflammation, have attracted attention as potential therapeutic targets in aging. On the other hand, there are few reports on the aging of the intestinal immune system, which differs from the systemic immune system in many ways. In the current study, we investigated the age-related changes in the intestinal immune system, particularly in T cells. The most significant changes were observed in the CD4+ T cells in the small intestinal IEL, with a marked increase in this fraction in old mice and reduced expression of CD27 and CD28, which are characteristic of aging systemic T cells. The proliferative capacity of aging IEL CD4+ T cells was significantly more reduced than that of aging systemic T cells. Transcriptome analysis showed that the expression of inflammatory cytokines was not upregulated, whereas Cd8α, NK receptors, and Granzymes were upregulated in aging IEL CD4+ T cells. Functional analysis showed that aging IEL T cells had a higher cytotoxic function against intestinal tumor organoids in vitro than young IEL T cells. scRNAseq revealed that splenic T cells show a transition from naïve to memory T cells, whereas intestinal T cells show the emergence of a CD8αα+CD4+ T cell fraction in aged mice, which is rarely seen in young cells. Further analysis of the aging IEL CD4+ T cells showed that two unique subsets are increased that are distinct from the systemic CD4+ T cells. Subset 1 has a pro-inflammatory component, with expression of IFNγ and upregulation of NFkB signaling pathways. Subset 2 does not express IFNγ, but upregulates inhibitory molecules and nIEL markers. Expression of granzymes and Cd8a was common to both. These fractions were in opposite positions in the clustering by UMAP and had different TCR repertoires. They may be involved in the suppression of intestinal aging and longevity through anti-tumor immunity, elimination of senescent cells and stressed cells in the aging environment. This finding could be a breakthrough in aging research.

Impact of prolonged cardiopulmonary resuscitation on outcomes in heart transplantation with higher risk donor heart.

OBJECTIVES: To evaluate the influence of prolonged cardiopulmonary resuscitation (CPR) on outcomes in heart transplantation with higher risk donor hearts (HRDHs). METHODS: Patients transplanted in our hospital between May 2006 and December 2019 were divided into 2 groups, HRDH recipients and non HRDH recipients. HRDH was defined as meeting at least one of the following criteria: (1) donor left ventricular ejection fraction ≤ 50%, (2) donor-recipient predicted heart mass ratio < 0.8 or > 1.2, (3) donor age ≥ 55 years, (4) ischemic time > 4 h and (5) catecholamine index > 20. Recipients of HRDHs were divided into 3 groups according to the time of CPR (Group1: non-CPR, Group 2: less than 30 min-CPR, and Group 3: longer than 30 min CPR). RESULTS: A total of 125 recipients were enrolled in this study, composing of HRDH recipients (n = 97, 78%) and non HRDH recipients (n = 28, 22%). Overall survival and the rate of freedom from cardiac events at 10 years after heart transplantation were comparable between two groups. Of 97 HRDH recipients, 54 (56%) without CPR, 22 (23%) with CPR < 30 min, and 21 (22%) with CPR ≥ 30 min were identified. One-year survival rates were not significantly different among three groups. The 1-year rate of freedom from cardiac events was not also statistically different, excluding the patients with coronary artery disease found in early postoperative period, which was thought to be donor-transmitted disease. Multivariate logistics regression for cardiac events identified that the CPR duration was not a risk factor even in HRDH-recipients. CONCLUSION: The CPR duration did not affect the outcomes after heart transplantation in HRDH recipients.

Spontaneous splenic rupture due to occult signet-ring cell gastric cancer accompanied by a bulky splenic vein thrombosis and postoperative brain infarction: A case report.

Background: Splenic rupture because of metastasis from a distant organ is extremely rare. Case Presentation: An 80-year-old man presented with left flank pain. A computed tomography (CT) demonstrated a poorly enhanced enlarged spleen with bulky thrombus in the splenic vein without extravasations. A CT on the following day showed increased intraperitoneal hemorrhage; therefore, an emergency laparotomy was performed. The spleen was enlarged and ruptured with lacerations on its surface. Macroscopic examination showed congestion with a thrombus in the splenic vein around the hilum. Pathology revealed signet-ring cell carcinoma. On the third postoperative day, a massive cerebral infarction in the left middle cerebral artery was revealed. Endoscopic examination demonstrated normal gastric mucosa except for some erosions, for which biopsies were performed, and two of five specimens encompassed signet-ring cell carcinoma in the lamina propria. Conclusion: Occult cancer could result in a drastic manifestation of its metastasis accompanying systemic thrombotic events.

Organoids transplantation as a new modality to design epithelial signature to create a membrane-protective sulfomucin-enriched segment.

BACKGROUND: The organoids therapy for ulcerative colitis (UC) is under development. It is important to dissect how the engrafted epithelium can provide benefits for overcoming the vulnerability to inflammation. We mainly focused on the deliverability of sulfomucin, which is reported to play an important role in epithelial function. METHODS: We analyzed each segment of colon epithelium to determine differences in sulfomucin production in both mice and human. Subsequently, we transplanted organoids established from sulfomucin-enriched region into the injured recipient epithelium following dextran sulfate sodium-induced colitis and analyzed the engrafted epithelium in mouse model. RESULTS: In human normal colon, sulfomucin production was increased in proximal colon, whereas it was decreased in the inflammatory region of UC. In murine colon epithelium, increased sulfomucin production was found in cecum compared to distal small intestine and proximal colon. RNA sequencing analysis revealed that several key genes associated with sulfomucin production such as Papss2 and Slc26a1 were enriched in isolated murine cecum crypts. Then we established murine cecum organoids and transplanted them into the injured epithelium of distal colon. Although the expression of sulfomucin was temporally decreased in cecum organoids, its secretion was restored again in the engrafted patches after transplantation. Finally, we verified a part of mechanisms controlling sulfomucin production in human samples. CONCLUSION: This study illustrated the deliverability of sulfomucin in the disease-relevant grafting model to design sulfomucin-producing epithelial units in severely injured distal colon. The current study is the basis for the better promotion of organoids transplantation therapy for refractory UC.

Collagen type I-mediated mechanotransduction controls epithelial cell fate conversion during intestinal inflammation.

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.

DNA methyltransferase 3A controls intestinal epithelial barrier function and regeneration in the colon.

Genetic variants in the DNA methyltransferase 3 A (DNMT3A) locus have been associated with inflammatory bowel disease (IBD). DNMT3A is part of the epigenetic machinery physiologically involved in DNA methylation. We show that DNMT3A plays a critical role in maintaining intestinal homeostasis and gut barrier function. DNMT3A expression is downregulated in intestinal epithelial cells from IBD patients and upon tumor necrosis factor treatment in murine intestinal organoids. Ablation of DNMT3A in Caco-2 cells results in global DNA hypomethylation, which is linked to impaired regenerative capacity, transepithelial resistance and intercellular junction formation. Genetic deletion of Dnmt3a in intestinal epithelial cells (Dnmt3aΔIEC) in mice confirms the phenotype of an altered epithelial ultrastructure with shortened apical-junctional complexes, reduced Goblet cell numbers and increased intestinal permeability in the colon in vivo. Dnmt3aΔIEC mice suffer from increased susceptibility to experimental colitis, characterized by reduced epithelial regeneration. These data demonstrate a critical role for DNMT3A in orchestrating intestinal epithelial homeostasis and response to tissue damage and suggest an involvement of impaired epithelial DNMT3A function in the etiology of IBD.

Zranb1-mutant mice display abnormal colonic mucus production and exacerbation of DSS-induced colitis.

Expression of mucin MUC2, a component of the colonic mucus layer, plays a crucial role in intestinal homeostasis. Here, we describe a new regulator of MUC2 expression, the deubiquitinase ZRANB1 (Trabid). A ZRANB1 mutation changing cysteine to serine in amino acid position 443, affects ubiquitination. To analyze ZRANB1 function in the intestine, we generated Zranb1 C443S mutant knock-in (Zranb1C443S/C443S) mice using the CRISPR/Cas9 system. Zranb1C443S/C443S mice exhibited decreased mRNA expression and MUC2 production. Colonic organoids from Zranb1C443S/C443S mice displayed decreased Muc2 mRNA expression following differentiation into goblet cells. Finally, we analyzed dextran sulfate sodium-induced colitis to understand ZRANB1's role in intestinal inflammation. Zranb1C443S/C443S mice with colitis exhibited significant weight loss, reduced colon length, and worsening clinical and pathological scores, indicating that ZRANB1 contributes to intestinal homeostasis. Together, these results suggest that ZRANB1 regulates MUC2 expression and intestinal inflammation, which may help elucidating the pathogenesis of inflammatory bowel disease and developing new therapeutics targeting ZRANB1.

Physiological role of anticipatory cardiorespiratory responses to exercise.

This study aimed to investigate whether anticipatory cardiorespiratory responses vary depending on the intensity of the subsequent exercise bout, and whether anticipatory cardiorespiratory adjustments contribute importantly to enhancing exercise performance during high-intensity exercise. Eleven healthy men were provided advance notice of the exercise intensity and a countdown to generate anticipation during 10 min prior to exercise at 0, 50, 80 or 95% maximal work-rate (Experiment 1). A different group of subjects (n = 15) performed a time to exhaustion trial with or without anticipatory countdown (Experiment 2). In Experiment 1, heart rate (HR), oxygen uptake (VO2 ) and minute ventilation (VE ) during pre-exercise resting period increased over time and depended on the subsequent exercise intensity. Specifically, there was already a 7.4% increase in HR from more than 5 min prior to the start of exercise at 95% maximal work-rate, followed by progressively augmented increases of 12.5% between 2 and 3 min before exercise, 24.4% between 0 and 1 min before exercise. In Experiment 2, the initial HR for the first 10 s of exercise in the task with anticipation was 11.4% larger compared to without anticipation (p < 0.01), and the difference in HR between the two conditions decreased in a time-dependent manner. In contrast, the initial increases in VO2 and VE were significantly lower in the task with anticipation than that without anticipation. The time to exhaustion during high-intensity exercise was 14.6% longer under anticipation condition compared to no anticipation (135 ± 26 s vs. 119 ± 26 s, p = 0.003). In addition, the enhanced exercise performance correlated positively with increased HR response just before and immediately after exercise onset (p < 0.01). These results showed that anticipatory cardiorespiratory adjustments (feedforward control) via the higher brain that operate before starting exercise may play an important role in minimizing the time delay of circulatory response and enhancing performance after onset of high-intensity exercise in man.

Retained intravascular catheter fragment at removal of implantable vascular access device: Incidence, risk factors, and outcomes.

BACKGROUND: Retained intravascular catheter fragments (RICF) are a rare complication of implantable vascular access device (IVAD) removal for which there is limited understanding of aetiology. There is a varied approach to management among the currently published literature. AIMS: The aim of this study was to establish incidence, risk factors, and outcomes for RICF after attempted removal of IVADs. METHODS: A single institution retrospective review was undertaken of individuals ≤ 25 years undergoing removal of IVADs from October 2014 to June 2019. Risk factors for RICF were explored using univariable logistic and Cox regression analysis. RESULTS: Six cases of RICF were identified among 654 line removal episodes (0.92% (95% CI 0.37-2%)) in patients aged 6-17 years (median 11, IQR 6-15 years). The main risk factor for RICF at removal was found to be line duration (OR 3.5/year, 95% CI 2.1-5.84, p < 0.0001). No RICFs occurred in lines indwelling for < 3 years. Five children with RICF ≤ 16 years were discussed with a paediatric cardiothoracic centre, and all were left in situ with 4 remaining asymptomatic. One had the fragment tip extruded through a wound, which required trimming. The other (17 years of age) developed an infected sinus for which partial removal with open excision followed by full removal with endovascular snare retrieval was performed by the adult vascular surgeons. CONCLUSION: IVADs in-situ for longer than a three-year period are at greatest risk of RICF upon removal. Management with transfixion of line fragments to surrounding muscle seems prudent while invasive attempts at retrieval appear unwarranted.

Epithelial X-Box Binding Protein 1 Coordinates Tumor Protein p53-Driven DNA Damage Responses and Suppression of Intestinal Carcinogenesis.

BACKGROUND & AIMS: Throughout life, the intestinal epithelium undergoes constant self-renewal from intestinal stem cells. Together with genotoxic stressors and failing DNA repair, this self-renewal causes susceptibility toward malignant transformation. X-box binding protein 1 (XBP1) is a stress sensor involved in the unfolded protein response (UPR). We hypothesized that XBP1 acts as a signaling hub to regulate epithelial DNA damage responses. METHODS: Data from The Cancer Genome Atlas were analyzed for association of XBP1 with colorectal cancer (CRC) survival and molecular interactions between XBP1 and p53 pathway activity. The role of XBP1 in orchestrating p53-driven DNA damage response was tested in vitro in mouse models of chronic intestinal epithelial cell (IEC) DNA damage (Xbp1/H2bfl/fl, Xbp1ΔIEC, H2bΔIEC, H2b/Xbp1ΔIEC) and via orthotopic tumor organoid transplantation. Transcriptome analysis of intestinal organoids was performed to identify molecular targets of Xbp1-mediated DNA damage response. RESULTS: In The Cancer Genome Atlas data set of CRC, low XBP1 expression was significantly associated with poor overall survival and reduced p53 pathway activity. In vivo, H2b/Xbp1ΔIEC mice developed spontaneous intestinal carcinomas. Orthotopic tumor organoid transplantation revealed a metastatic potential of H2b/Xbp1ΔIEC-derived tumors. RNA sequencing of intestinal organoids (H2b/Xbp1fl/fl, H2bΔIEC, H2b/Xbp1ΔIEC, and H2b/p53ΔIEC) identified a transcriptional program downstream of p53, in which XBP1 directs DNA-damage-inducible transcript 4-like (Ddit4l) expression. DDIT4L inhibits mechanistic target of rapamycin-mediated phosphorylation of 4E-binding protein 1. Pharmacologic mechanistic target of rapamycin inhibition suppressed epithelial hyperproliferation via 4E-binding protein 1. CONCLUSIONS: Our data suggest a crucial role for XBP1 in coordinating epithelial DNA damage responses and stem cell function via a p53-DDIT4L-dependent feedback mechanism.

Genome-wide analysis of 944 133 individuals provides insights into the etiology of haemorrhoidal disease.

OBJECTIVE: Haemorrhoidal disease (HEM) affects a large and silently suffering fraction of the population but its aetiology, including suspected genetic predisposition, is poorly understood. We report the first genome-wide association study (GWAS) meta-analysis to identify genetic risk factors for HEM to date. DESIGN: We conducted a GWAS meta-analysis of 218 920 patients with HEM and 725 213 controls of European ancestry. Using GWAS summary statistics, we performed multiple genetic correlation analyses between HEM and other traits as well as calculated HEM polygenic risk scores (PRS) and evaluated their translational potential in independent datasets. Using functional annotation of GWAS results, we identified HEM candidate genes, which differential expression and coexpression in HEM tissues were evaluated employing RNA-seq analyses. The localisation of expressed proteins at selected loci was investigated by immunohistochemistry. RESULTS: We demonstrate modest heritability and genetic correlation of HEM with several other diseases from the GI, neuroaffective and cardiovascular domains. HEM PRS validated in 180 435 individuals from independent datasets allowed the identification of those at risk and correlated with younger age of onset and recurrent surgery. We identified 102 independent HEM risk loci harbouring genes whose expression is enriched in blood vessels and GI tissues, and in pathways associated with smooth muscles, epithelial and endothelial development and morphogenesis. Network transcriptomic analyses highlighted HEM gene coexpression modules that are relevant to the development and integrity of the musculoskeletal and epidermal systems, and the organisation of the extracellular matrix. CONCLUSION: HEM has a genetic component that predisposes to smooth muscle, epithelial and connective tissue dysfunction.

Notch and TNF-α signaling promote cytoplasmic accumulation of OLFM4 in intestinal epithelium cells and exhibit a cell protective role in the inflamed mucosa of IBD patients.

Notch signaling is activated in the intestinal epithelial cells (IECs) of patients with inflammatory bowel disease (IBD), and contributes to mucosal regeneration. Our previous study indicated that TNF-α and Notch signaling may synergistically promote the expression of the intestinal stem cell (ISC) marker OLFM4 in human IECs. In the present study, we investigated the gene regulation and function of OLFM4 in human IEC lines. We confirmed that TNF-α and Notch synergistically upregulate the mRNA expression of OLFM4. Luciferase reporter assay showed that OLFM4 transcription is regulated by the synergy of TNF-α and Notch. At the protein level, synergy between TNF-α and Notch promoted cytoplasmic accumulation of OLFM4, which has potential anti-apoptotic properties in human IECs. Analysis of patient-derived tissues and organoids consistently showed cytoplasmic accumulation of OLFM4 in response to NF-κB and Notch activation. Cytoplasmic accumulation of OLFM4 in human IECs is tightly regulated by Notch and TNF-α in synergy. Such cytoplasmic accumulation of OLFM4 may have a cell-protective role in the inflamed mucosa of patients with IBD.

Functional analysis of isoflavones using patient-derived human colonic organoids.

Inflammatory bowel disease (IBD) comprises two major subtypes, ulcerative colitis (UC) and Crohn's disease, which are multifactorial diseases that may develop due to genetic susceptibility, dysbiosis, or environmental factors. Environmental triggers of IBD include food-borne factors, and a previous nationwide survey in Japan identified pre-illness consumption of isoflavones as a risk factor for UC. However, the precise mechanisms involved in the detrimental effects of isoflavones on the intestinal mucosa remain unclear. The present study employed human colonic organoids (hCOs) to investigate the functional effect of two representative isoflavones, genistein and daidzein, on human colonic epithelial cells. The addition of genistein to organoid reformation assays significantly decreased the number and size of reformed hCOs compared with control and daidzein treatment, indicating an inhibitory effect of genistein on colonic cell/progenitor cell function. Evaluation of the phosphorylation status of 49 different receptor tyrosine kinases showed that genistein selectively inhibited phosphorylation of epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (HGFR). We established a two-dimensional wound-repair model using hCOs and showed that genistein significantly delayed the overall wound-repair response. Our results collectively show that genistein may exert its detrimental effects on the intestinal mucosa via negative regulation of stem/progenitor cell function, possibly leading to sustained mucosal injury and the development of UC.

Activating Transcription Factor 6 Mediates Inflammatory Signals in Intestinal Epithelial Cells Upon Endoplasmic Reticulum Stress.

BACKGROUND & AIMS: Excess and unresolved endoplasmic reticulum (ER) stress in intestinal epithelial cells (IECs) promotes intestinal inflammation. Activating transcription factor 6 (ATF6) is one of the signaling mediators of ER stress. We studied the pathways that regulate ATF6 and its role for inflammation in IECs. METHODS: We performed an RNA interference screen, using 23,349 unique small interfering RNAs targeting 7783 genes and a luciferase reporter controlled by an ATF6-dependent ERSE (ER stress-response element) promoter, to identify proteins that activate or inhibit the ATF6 signaling pathway in HEK293 cells. To validate the screening results, intestinal epithelial cell lines (Caco-2 cells) were transfected with small interfering RNAs or with a plasmid overexpressing a constitutively active form of ATF6. Caco-2 cells with a CRISPR-mediated disruption of autophagy related 16 like 1 gene (ATG16L1) were used to study the effect of ATF6 on ER stress in autophagy-deficient cells. We also studied intestinal organoids derived from mice that overexpress constitutively active ATF6, from mice with deletion of the autophagy related 16 like 1 or X-Box binding protein 1 gene in IECs (Atg16l1ΔIEC or Xbp1ΔIEC, which both develop spontaneous ileitis), from patients with Crohn's disease (CD) and healthy individuals (controls). Cells and organoids were incubated with tunicamycin to induce ER stress and/or chemical inhibitors of newly identified activator proteins of ATF6 signaling, and analyzed by real-time polymerase chain reaction and immunoblots. Atg16l1ΔIEC and control (Atg16l1fl/fl) mice were given intraperitoneal injections of tunicamycin and were treated with chemical inhibitors of ATF6 activating proteins. RESULTS: We identified and validated 15 suppressors and 7 activators of the ATF6 signaling pathway; activators included the regulatory subunit of casein kinase 2 (CSNK2B) and acyl-CoA synthetase long chain family member 1 (ACSL1). Knockdown or chemical inhibition of CSNK2B and ACSL1 in Caco-2 cells reduced activity of the ATF6-dependent ERSE reporter gene, diminished transcription of the ATF6 target genes HSP90B1 and HSPA5 and reduced NF-κB reporter gene activation on tunicamycin stimulation. Atg16l1ΔIEC and or Xbp1ΔIEC organoids showed increased expression of ATF6 and its target genes. Inhibitors of ACSL1 or CSNK2B prevented activation of ATF6 and reduced CXCL1 and tumor necrosis factor (TNF) expression in these organoids on induction of ER stress with tunicamycin. Injection of mice with inhibitors of ACSL1 or CSNK2B significantly reduced tunicamycin-mediated intestinal inflammation and IEC death and expression of CXCL1 and TNF in Atg16l1ΔIEC mice. Purified ileal IECs from patients with CD had higher levels of ATF6, CSNK2B, and HSPA5 messenger RNAs than controls; early-passage organoids from patients with active CD show increased levels of activated ATF6 protein, incubation of these organoids with inhibitors of ACSL1 or CSNK2B reduced transcription of ATF6 target genes, including TNF. CONCLUSIONS: Ileal IECs from patients with CD have higher levels of activated ATF6, which is regulated by CSNK2B and HSPA5. ATF6 increases expression of TNF and other inflammatory cytokines in response to ER stress in these cells and in organoids from Atg16l1ΔIEC and Xbp1ΔIEC mice. Strategies to inhibit the ATF6 signaling pathway might be developed for treatment of inflammatory bowel diseases.

Dynamic characteristics of cerebrovascular reactivity or ventilatory response to change in carbon dioxide.

NEW FINDINGS: What is the central question of this study? What are the dynamic characteristics of cerebrovascular carbon dioxide reactivity and the central respiratory chemoreflex? What is the main finding and its importance? The transfer function gain from the end-tidal partial pressure of carbon dioxide to cerebral blood flow or ventilation decreased in the high frequency range at rest and during exercise. These findings indicate that the dynamic characteristics of both systems were not constant in all frequency ranges, and this trend was not modified by exercise. ABSTRACT: The purpose of this study was to examine the dynamic characteristics of cerebrovascular reactivity and ventilatory response to change in arterial CO2 in all frequency ranges at rest using frequency domain analysis, and also to examine whether this is modified by dynamic exercise as with the traditionally determined cerebrovascular CO2 reactivity. In nine healthy young subjects, at rest and during exercise (cycling exercise at constant predetermined work rate corresponding to a V̇O2 level of 0.90 l min-1 ), the dynamic characteristics of cerebrovascular CO2 reactivity and the central respiratory chemoreflex were assessed by transfer function analysis using a binary white-noise sequence (0-7% inspired CO2 fraction) from the end-tidal partial pressure of CO2 ( PETCO2 ) to the mean middle cerebral artery mean blood velocity (MCA Vm ) or minute ventilation ( V̇E ), respectively. In the high frequency range, both transfer function gains decreased but, interestingly, the cut-off frequency in the transfer function gain from PETCO2 to MCA Vm response was higher than that from PETCO2 to V̇E response at rest (0.024 vs. 0.015 Hz) and during exercise (0.030 vs. 0.011 Hz), indicating that cerebrovascular CO2 reactivity or central respiratory chemoreflex was not constant in all frequency ranges, and this trend was not modified by exercise. These findings suggest that dynamic characteristics of the cerebrovascular CO2 reactivity or central chemoreflex need to be assessed to identify the whole system because the traditional method cannot identify the property of time response of these systems.

Depletion of erythropoietic miR-486-5p and miR-451a improves detectability of rare microRNAs in peripheral blood-derived small RNA sequencing libraries.

Erythroid-specific miR-451a and miR-486-5p are two of the most dominant microRNAs (miRNAs) in human peripheral blood. In small RNA sequencing libraries, their overabundance reduces diversity as well as complexity and consequently causes negative effects such as missing detectability and inaccurate quantification of low abundant miRNAs. Here we present a simple, cost-effective and easy to implement hybridization-based method to deplete these two erythropoietic miRNAs from blood-derived RNA samples. By utilization of blocking oligonucleotides, this method provides a highly efficient and specific depletion of miR-486-5p and miR-451a, which leads to a considerable increase of measured expression as well as detectability of low abundant miRNA species. The blocking oligos are compatible with common 5' ligation-dependent small RNA library preparation protocols, including commercially available kits, such as Illumina TruSeq and Perkin Elmer NEXTflex. Furthermore, the here described method and oligo design principle can be easily adapted to target many other miRNA molecules, depending on context and research question.