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.

Canthin-6-One Inhibits Developmental and Tumour-Associated Angiogenesis in Zebrafish.

Tumour-associated angiogenesis play key roles in tumour growth and cancer metastasis. Consequently, several anti-angiogenic drugs such as sunitinib and axitinib have been approved for use as anti-cancer therapies. However, the majority of these drugs target the vascular endothelial growth factor A (VEGFA)/VEGF receptor 2 (VEGFR2) pathway and have shown mixed outcome, largely due to development of resistances and increased tumour aggressiveness. In this study, we used the zebrafish model to screen for novel anti-angiogenic molecules from a library of compounds derived from natural products. From this, we identified canthin-6-one, an indole alkaloid, which inhibited zebrafish intersegmental vessel (ISV) and sub-intestinal vessel development. Further characterisation revealed that treatment of canthin-6-one reduced ISV endothelial cell number and inhibited proliferation of human umbilical vein endothelial cells (HUVECs), suggesting that canthin-6-one inhibits endothelial cell proliferation. Of note, canthin-6-one did not inhibit VEGFA-induced phosphorylation of VEGFR2 in HUVECs and downstream phosphorylation of extracellular signal-regulated kinase (Erk) in leading ISV endothelial cells in zebrafish, suggesting that canthin-6-one inhibits angiogenesis independent of the VEGFA/VEGFR2 pathway. Importantly, we found that canthin-6-one impairs tumour-associated angiogenesis in a zebrafish B16F10 melanoma cell xenograft model and synergises with VEGFR inhibitor sunitinib malate to inhibit developmental angiogenesis. In summary, we showed that canthin-6-one exhibits anti-angiogenic properties in both developmental and pathological contexts in zebrafish, independent of the VEGFA/VEGFR2 pathway and demonstrate that canthin-6-one may hold value for further development as a novel anti-angiogenic drug.

Vasculature is getting Hip(po): Hippo signaling in vascular development and disease.

The Hippo signaling pathway regulates developmental organ growth, regeneration, and cell fate decisions. Although the role of the Hippo pathway, and its transcriptional effectors YAP and TAZ, has been well documented in many cell types and species, only recently have the roles for this pathway come to light in vascular development and disease. Experiments in mice, zebrafish, and in vitro have uncovered roles for the Hippo pathway, YAP, and TAZ in vasculogenesis, angiogenesis, and lymphangiogenesis. In addition, the Hippo pathway has been implicated in vascular cancers and cardiovascular diseases, thus identifying it as a potential therapeutic target for the treatment of these conditions. However, despite recent advances, Hippo's role in the vasculature is still underappreciated compared with its role in epithelial tissues. In this review, we appraise our current understanding of the Hippo pathway in blood and lymphatic vessel development and highlight the current knowledge gaps and opportunities for further research.

Single-cell analysis of lymphatic endothelial cell fate specification and differentiation during zebrafish development.

During development, the lymphatic vasculature forms as a second network derived chiefly from blood vessels. The transdifferentiation of embryonic venous endothelial cells (VECs) into lymphatic endothelial cells (LECs) is a key step in this process. Specification, differentiation and maintenance of LEC fate are all driven by the transcription factor Prox1, yet the downstream mechanisms remain to be elucidated. We here present a single-cell transcriptomic atlas of lymphangiogenesis in zebrafish, revealing new markers and hallmarks of LEC differentiation over four developmental stages. We further profile single-cell transcriptomic and chromatin accessibility changes in zygotic prox1a mutants that are undergoing a LEC-VEC fate shift. Using maternal and zygotic prox1a/prox1b mutants, we determine the earliest transcriptomic changes directed by Prox1 during LEC specification. This work altogether reveals new downstream targets and regulatory regions of the genome controlled by Prox1 and presents evidence that Prox1 specifies LEC fate primarily by limiting blood vascular and haematopoietic fate. This extensive single-cell resource provides new mechanistic insights into the enigmatic role of Prox1 and the control of LEC differentiation in development.

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.

The bactericidal and biofilm removal effect of super reducing water on Streptococcus mutans in three types of orthodontic brackets.

PURPOSE: To investigate the bactericidal and biofilm removal effect of super reducing water (SRW) on Streptococcus mutans (S. mutans) adhered to orthodontic brackets, in vitro. METHODS: Three types of brackets were bonded to aluminum disks. After the formation of S. mutans biofilms on the surfaces, the brackets were divided into three groups (n = 44 each) based on their exposure to SRW: group 1, no treatment; group 2, treated for 5 min; and group 3, treated for 10 min. Total viable counts, adenosine triphosphate measurements, crystal violet assay, and scanning electron microscopy were used to evaluate the effect of SRW. RESULTS: The bacterial counts in groups 2 and 3 were significantly lower than those in group 1 (P < 0.001); however, no significant differences were observed between groups 2 and 3. Marked decreases in the number of bacterial colonies and extent of biofilm formation were observed in groups 2 and 3 compared to group 1. No significant differences in the number of bacterial colonies and amount of biofilm were observed among the three types of brackets in each group. CONCLUSION: These findings indicate the bactericidal and biofilm removal effect of SRW treatment on S. mutans adhered to orthodontic brackets.

Influence of mandibular incisor agenesis and growth pattern on symphysis characteristics: A retrospective cephalometric study.

OBJECTIVE: This study was performed to investigate the effects of mandibular incisor (MnI) agenesis and divergent malocclusion type on mandibular symphysis inclination and morphology. METHODS: A total of 162 selected patients were divided into two groups: one group consisted of patients with one or two congenitally missing MnIs, and another group comprised patients without tooth agenesis. Patients in each group were categorized into three divergent malocclusion groups (hypodivergent, normodivergent and hyperdivergent) according to the Frankfort mandibular plane angle, with 27 patients per group. Lateral cephalograms were used to evaluate mandibular symphysis inclination and morphology. Two-way analysis of variance, simple main effect analysis and Tukey's test were used for statistical comparisons. RESULTS: The agenesis group demonstrated a significantly greater retroclination of the mandibular symphysis than the non-agenesis group in the normodivergent group. In the hypodivergent and normodivergent groups, the agenesis group showed a significantly smaller area of the alveolar bone with thinner width and shorter height than the non-agenesis group. CONCLUSION: For the Japanese orthodontic patients, MnI agenesis caused a significantly great retroclination of the mandibular symphysis in patients with normodivergent malocclusion and significantly small area of the alveolar bone with thin width and short height in patients with hypo- and normodivergent malocclusions.

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.

Transplantation of intestinal organoids into a mouse model of colitis.

Intestinal organoids are fundamental in vitro tools that have enabled new research opportunities in intestinal stem cell research. Organoids can also be transplanted in vivo, which enables them to probe stem cell potential and be used for disease modeling and as a preclinical tool in regenerative medicine. Here we describe in detail how to orthotopically transplant epithelial organoids into the colon of recipient mice. In this assay, epithelial injury is initiated at the distal part of colon by the administration of dextran sulfate sodium, and organoids are infused into the luminal space via the anus. The infused organoids subsequently attach to the injured region and rebuild a donor-derived epithelium. The steps for cell infusion can be completed in 10 min. The assay has been applied successfully to organoids derived from both wild-type and genetically altered epithelial cells from adult colonic and small intestinal epithelium, as well as fetal small intestine. This is a versatile protocol, providing the technical basis for transplantation following alternative colonic injury models. It has been used previously for functional assays to probe cellular potential, and formed the basis for the first in-human clinical trial using colonic organoid transplantation therapy for intractable cases of ulcerative colitis.

TGF-ß promotes fetal gene expression and cell migration velocity in a wound repair model of untransformed intestinal epithelial cells.

The early-phase wound repair response of the intestinal epithelium is characterized by rapid and organized cell migration. This response is regulated by several humoral factors, including TGF-ß. However, due to a lack of appropriate models, the precise response of untransformed intestinal epithelial cells (IECs) to those factors is unclear. In this study, we established an in vitro wound repair model of untransformed IECs, based on native type-I collagen. In our system, IECs formed a uniform monolayer in a two-chamber culture insert and displayed a stable wound repair response. Gene expression analysis revealed significant induction of Apoa1, Apoa4, and Wnt4 during the collagen-guided wound repair response. The wound repair response was enhanced significantly by the addition of TGF-ß. Surprisingly, addition of TGF-ß induced a set of genes, including Slc28a2, Tubb2a, and Cpe, that were expressed preferentially in fetal IECs. Moreover, TGF-ß significantly increased the peak velocity of migrating IECs and, conversely, reduced the time required to reach the peak velocity, as confirmed by the motion vector prediction (MVP) method. Our current in vitro system could be employed to assess other humoral factors involved in IEC migration and could contribute to a deeper understanding of the wound repair potentials of untransformed IECs.

Refractory Chylothorax and Lymphedema Caused by Advanced Gastric Cancer.

Chylothorax is the accumulation of lipid pleural effusion. Few reports have described chylothorax caused by gastric cancer. A 45-year-old woman presented with progressive lymphedema and bilateral chylothorax. Although repetitive thoracentesis was performed to relieve her dyspnea, swelling of her axillary lymph nodes became significant. Positron emission tomography/computed tomography demonstrated the accumulation of 18F-fluorodeoxyglucose in these nodes, and a lymph node biopsy showed signet ring cell carcinoma. The primary site was a 0-IIc type lesion in the gastric body that was only detected by upper gastrointestinal endoscopy. The patient was diagnosed with advanced gastric cancer 3.5 months after presentation for chylothorax.

Next endoscopic approach for acute lower gastrointestinal bleeding without an identified source on colonoscopy: upper or capsule endoscopy?

Background and study aims We evaluated the utility of esophagogastroduodenoscopy (EGD) or capsule endoscopy (CE) as the next diagnostic approach after negative colonoscopy (CS) results in acute-onset hematochezia. Patients and methods We retrospectively analyzed 401 patients emergently hospitalized for acute hematochezia who underwent CS within 48 hours of arriving at two large emergency hospitals and in whom a definitive bleeding source was not identified. The positive endoscopic findings, requirement for additional therapeutic procedures, and 30-day rebleeding rates were compared among three strategies: EGD following CS (CS-EGD), CE following CS (CS-CE), and CS alone. Predictors of positive endoscopic findings in the CS-EGD strategy were determined. Results The rates of positive endoscopic findings and requirement for additional therapeutic procedures were 22 % and 16 %, respectively, in CS-EGD and 50 % and 28 % in CS-CE. The 30-day rebleeding rate did not significantly decrease in CS-EGD (8 %) or CS-CE (11 %) compared with CS alone (12 %). The rate of additional endoscopic therapies was lower in patients with a colonic diverticulum than in those without (CS-EGD: 3 % vs. 33 %, P  = 0.007; CS-CE: 11 % vs. 44 %, P  = 0.147). A history of syncope, low blood pressure, blood urea nitrogen/creatinine ratio of ≥ 30, and low albumin level significantly predicted EGD findings after negative CS results ( P  < 0.05). Conclusions When the definitive bleeding source is not identified by colonoscopy in patients with acute hematochezia, adjunctive endoscopy helps to identify the etiology and enables subsequent therapy, especially for patients without a colonic diverticulum. Upper gastrointestinal endoscopy is indicated for severe bleeding; other patients may be candidates for capsule endoscopy.

Post-polypectomy bleeding and thromboembolism risks associated with warfarin vs direct oral anticoagulants.

AIM: To verify the validity of the endoscopy guidelines for patients taking warfarin or direct oral anticoagulants (DOAC). METHODS: We collected data from 218 patients receiving oral anticoagulants (73 DOAC users, 145 warfarin users) and 218 patients not receiving any antithrombotics (age- and sex-matched controls) who underwent polypectomy. (1) We evaluated post-polypectomy bleeding (PPB) risk in patients receiving warfarin or DOAC compared with controls; (2) we assessed the risks of PPB and thromboembolism between three AC management methods: Discontinuing AC with heparin bridge (HPB) (endoscopy guideline recommendation), continuing AC, and discontinuing AC without HPB. RESULTS: PPB rate was significantly higher in warfarin users and DOAC users compared with controls (13.7% and 13.7% vs 0.9%, P < 0.001), but was not significantly different between rivaroxaban (13.2%), dabigatran (11.1%), and apixaban (13.3%) users. Two thromboembolic events occurred in warfarin users, but none in DOAC users. Compared with the continuing anticoagulant group, the discontinuing anticoagulant with HPB group (guideline recommendation) had a higher PPB rate (10.8% vs 19.6%, P = 0.087). These findings were significantly evident in warfarin but not DOAC users. One thrombotic event occurred in the discontinuing anticoagulant with HPB group and the discontinuing anticoagulant without HPB group; none occurred in the continuing anticoagulant group. CONCLUSION: PPB risk was similar between patients taking warfarin and DOAC. Thromboembolism was observed in warfarin users only. The guideline recommendations for HPB should be re-considered.

Temperature-induced Coalescence of Droplets Manipulated by Optical Trapping in an Oil-in-Water Emulsion.

Coalescence of oil droplets in an oil-in-water (O/W) emulsion was achieved with heating and optical trapping. Three types of O/W emulsions were prepared by adding a mixture of butanol and n-decane to an aqueous solution containing a cationic surfactant (cetyltrimethylammonium bromide, CTAB), an anionic surfactant (sodium dodecyl sulfate, SDS), or a neutral hydrophilic polymer (polyethylene glycol, PEG) as an emulsifier. Two oil droplets in the emulsions were randomly trapped in a square capillary tube by two laser beams in order to induce coalescence. Coalescence of the droplets could not be achieved at room temperature (25°C) regardless of the type of emulsifier. Conversely, the droplets prepared with PEG coalesced at a temperature higher than 30°C, although the droplets with ionic surfactants CTAB and SDS did not coalesce even at the elevated temperature due to their electrostatic repulsion. The size of the resultant coalesced droplet was consistent with that calculated from the size of the two droplets of oil, which indicated successful coalescence of the two droplets. We also found that the time required for the coalescence could be correlated with the temperature using an Arrhenius plot.

Differentiation of oligodendrocyte progenitor cells from dissociated monolayer and feeder-free cultured pluripotent stem cells.

Oligodendrocytes myelinate axons and form myelin sheaths in the central nervous system. The development of therapies for demyelinating diseases, including multiple sclerosis and leukodystrophies, is a challenge because the pathogenic mechanisms of disease remain poorly understood. Primate pluripotent stem cell-derived oligodendrocytes are expected to help elucidate the molecular pathogenesis of these diseases. Oligodendrocytes have been successfully differentiated from human pluripotent stem cells. However, it is challenging to prepare large amounts of oligodendrocytes over a short amount of time because of manipulation difficulties under conventional primate pluripotent stem cell culture methods. We developed a proprietary dissociated monolayer and feeder-free culture system to handle pluripotent stem cell cultures. Because the dissociated monolayer and feeder-free culture system improves the quality and growth of primate pluripotent stem cells, these cells could potentially be differentiated into any desired functional cells and consistently cultured in large-scale conditions. In the current study, oligodendrocyte progenitor cells and mature oligodendrocytes were generated within three months from monkey embryonic stem cells. The embryonic stem cell-derived oligodendrocytes exhibited in vitro myelinogenic potency with rat dorsal root ganglion neurons. Additionally, the transplanted oligodendrocyte progenitor cells differentiated into myelin basic protein-positive mature oligodendrocytes in the mouse corpus callosum. This preparative method was used for human induced pluripotent stem cells, which were also successfully differentiated into oligodendrocyte progenitor cells and mature oligodendrocytes that were capable of myelinating rat dorsal root ganglion neurons. Moreover, it was possible to freeze, thaw, and successfully re-culture the differentiating cells. These results showed that embryonic stem cells and human induced pluripotent stem cells maintained in a dissociated monolayer and feeder-free culture system have the potential to generate oligodendrocyte progenitor cells and mature oligodendrocytes in vitro and in vivo. This culture method could be applied to prepare large amounts of oligodendrocyte progenitor cells and mature oligodendrocytes in a relatively short amount of time.

Gpr176 is a Gz-linked orphan G-protein-coupled receptor that sets the pace of circadian behaviour.

G-protein-coupled receptors (GPCRs) participate in a broad range of physiological functions. A priority for fundamental and clinical research, therefore, is to decipher the function of over 140 remaining orphan GPCRs. The suprachiasmatic nucleus (SCN), the brain's circadian pacemaker, governs daily rhythms in behaviour and physiology. Here we launch the SCN orphan GPCR project to (i) search for murine orphan GPCRs with enriched expression in the SCN, (ii) generate mutant animals deficient in candidate GPCRs, and (iii) analyse the impact on circadian rhythms. We thereby identify Gpr176 as an SCN-enriched orphan GPCR that sets the pace of circadian behaviour. Gpr176 is expressed in a circadian manner by SCN neurons, and molecular characterization reveals that it represses cAMP signalling in an agonist-independent manner. Gpr176 acts independently of, and in parallel to, the Vipr2 GPCR, not through the canonical Gi, but via the unique G-protein subclass Gz.