Epigenetic modifications underlie the differential adipogenic potential of preadipocytes derived from human subcutaneous fat tissue.

Ceiling culture-derived preadipocytes (ccdPAs) and adipose-derived stem cells (ASCs) can be harvested from human subcutaneous fat tissue using the specific gravity method. Both cell types possess a similar spindle shape without lipid droplets. We previously reported that ccdPAs have a higher adipogenic potential than ASCs, even after a 7-wk culture. We performed a genome-wide epigenetic analysis to examine the mechanisms contributing to the adipogenic potential differences between ccdPAs and ASCs. Methylation analysis of cytosines followed by guanine (CpG) using a 450-K BeadChip was performed on human ccdPAs and ASCs isolated from three metabolically healthy females. Chromatin immunoprecipitation sequencing was performed to evaluate trimethylation at lysine 4 of histone 3 (H3K4me3). Unsupervised machine learning using t-distributed stochastic neighbor embedding to interpret 450,000-dimensional methylation assay data showed that the cells were divided into ASC and ccdPA groups. In Kyoto Encyclopedia of Genes and Genomes pathway analysis of 1,543 genes with differential promoter CpG methylation, the peroxisome proliferator-activated receptor (PPAR) and adipocytokine signaling pathways ranked in the top 10 pathways. In the PPARγ gene, H3K4me3 peak levels were higher in ccdPAs than in ASCs, whereas promoter CpG methylation levels were significantly lower in ccdPAs than in ASCs. Similar differences in promoter CpG methylation were also seen in the fatty acid-binding protein 4 and leptin genes. In conclusion, we analyzed the epigenetic status of adipogenesis-related genes as a potential mechanism underlying the differences in adipogenic differentiation capability between ASCs and ccdPAs.

TAS4464, a NEDD8-activating enzyme inhibitor, activates both intrinsic and extrinsic apoptotic pathways via c-Myc-mediated regulation in acute myeloid leukemia.

TAS4464, a potent, selective small molecule NEDD8-activating enzyme (NAE) inhibitor, leads to inactivation of cullin-RING E3 ubiquitin ligases (CRLs) and consequent accumulations of its substrate proteins. Here, we investigated the antitumor properties and action mechanism of TAS4464 in acute myeloid leukemia (AML). TAS4464 induced apoptotic cell death in various AML cell lines. TAS4464 treatments resulted in the activation of both the caspase-9-mediated intrinsic apoptotic pathway and caspase-8-mediated extrinsic apoptotic pathway in AML cells; combined treatment with inhibitors of these caspases markedly diminished TAS4464-induced apoptosis. In each apoptotic pathway, TAS4464 induced the mRNA transcription of the intrinsic proapoptotic factor NOXA and decreased that of the extrinsic antiapoptotic factor c-FLIP. RNA-sequencing analysis showed that the signaling pathway of the CRL substrate c-Myc was enriched after TAS4464 treatment. Chromatin immunoprecipitation (ChIP) assay revealed that TAS4464-induced c-Myc bound to the PMAIP1 (encoding NOXA) and CFLAR (encoding c-FLIP) promoter regions, and siRNA-mediated c-Myc knockdown neutralized both TAS4464-mediated NOXA induction and c-FLIP downregulation. TAS4464 activated both caspase-8 and caspase-9 along with an increase in NOXA and a decrease in c-FLIP, resulting in complete tumor remission in a human AML xenograft model. These findings suggest that NAE inhibition leads to anti-AML activity via a novel c-Myc-dependent apoptosis induction mechanism.

Ascl2-Dependent Cell Dedifferentiation Drives Regeneration of Ablated Intestinal Stem Cells.

Ablation of LGR5+ intestinal stem cells (ISCs) is associated with rapid restoration of the ISC compartment. Different intestinal crypt populations dedifferentiate to provide new ISCs, but the transcriptional and signaling trajectories that guide this process are unclear, and a large body of work suggests that quiescent "reserve" ISCs contribute to regeneration. By timing the interval between LGR5+ lineage tracing and lethal injury, we show that ISC regeneration is explained nearly completely by dedifferentiation, with contributions from absorptive and secretory progenitors. The ISC-restricted transcription factor ASCL2 confers measurable competitive advantage to resting ISCs and is essential to restore the ISC compartment. Regenerating cells re-express Ascl2 days before Lgr5, and single-cell RNA sequencing (scRNA-seq) analyses reveal transcriptional paths underlying dedifferentiation. ASCL2 target genes include the interleukin-11 (IL-11) receptor Il11ra1, and recombinant IL-11 enhances crypt cell regenerative potential. These findings reveal cell dedifferentiation as the principal means for ISC restoration and highlight an ASCL2-regulated signal that enables this adaptive response.

Dynamic Reorganization of Chromatin Accessibility Signatures during Dedifferentiation of Secretory Precursors into Lgr5+ Intestinal Stem Cells.

Replicating Lgr5+ stem cells and quiescent Bmi1+ cells behave as intestinal stem cells (ISCs) in vivo. Disrupting Lgr5+ ISCs triggers epithelial renewal from Bmi1+ cells, from secretory or absorptive progenitors, and from Paneth cell precursors, revealing a high degree of plasticity within intestinal crypts. Here, we show that GFP+ cells from Bmi1GFP mice are preterminal enteroendocrine cells and we identify CD69+CD274+ cells as related goblet cell precursors. Upon loss of native Lgr5+ ISCs, both populations revert toward an Lgr5+ cell identity. While active histone marks are distributed similarly between Lgr5+ ISCs and progenitors of both major lineages, thousands of cis elements that control expression of lineage-restricted genes are selectively open in secretory cells. This accessibility signature dynamically converts to that of Lgr5+ ISCs during crypt regeneration. Beyond establishing the nature of Bmi1GFP+ cells, these findings reveal how chromatin status underlies intestinal cell diversity and dedifferentiation to restore ISC function and intestinal homeostasis.

Development of electrolyte-free ozone sensors using boron-doped diamond electrodes.

The electrochemical detection of dissolved ozone in water was examined using boron-doped diamond (BDD) electrodes. A well-defined reduction peak was observed at ~380 mV for H-terminated BDD, whereas it was observed at ~200 mV in the case of O-terminated BDD for an ozone solution in a 0.1 M phosphate buffer solution at pH 7. The peak potential for ozone reduction was selective with respect to oxygen reduction at both H- and O-terminated BDD electrodes, whereas it occurred at approximately the same potential as oxygen reduction at other types of solid electrodes, including glassy-carbon, platinum, and gold electrodes. Interference from chlorine was not observed in lower concentration than 300 µM ClO(-). Furthermore, in order to apply the detection technique to electrolyte-free media, BDD microelectrodes were also used. A linear calibration curve for dissolved ozone in water could be achieved between concentrations of 0.49 and 740 µM, with an estimated detection limit (S/N = 3) of 0.185 µM (S/N = 3). Excellent stability was demonstrated for repetitions of these calibration curves performed in 3 consecutive days.

Proliferation and harvest of human mesenchymal stem cells using new thermoresponsive nanocomposite gels.

For tissue engineering and regenerative medicine, stem cells should be effectively cultured in vitro. New thermoresponsive nanocomposite gels (MD-NC gels), consisting of inorganic clay (hectorite) and copolymers composed of hydrophobic 2-methoxyethyl acrylate (MEA) and hydrophilic N,N-dimethylacrylamide (DMAA) units, could be applied in cell culture and cell harvesting without trypsinization, specifically using mesenchymal stem cells (MSCs). The composition of the MD-NC gel (the ratio of the two monomer types and the clay content) was found to determine its swelling properties in the culture medium, thermosensitivity, protein adsorption, and cell attachment and proliferation. Various kinds of human cells, including MSCs, osteoblast (HOS) cells, fibroblast (NHDF) cells, and epithelial cells could be effectively cultured on MD-NC gels. In particular, on an MD10-NC2 gel with relatively low DMAA and clay content, the cells could be harvested by decreasing the temperature, either as a cell sheet (MSCs or NHDF cells) or as a population of suspension cells (HOS cells). Further, it was found that the MD10-NC2 gel is suitable for stem cell differentiation. Because of their thermosensitivity, controllable modulus, and surface properties, MD-NC gels are promising cell culture substrates useful for tissue engineering and regenerative medicine.

Randomized controlled trial comparing antioxidant-enriched enteral nutrition with immune-enhancing enteral nutrition after esophagectomy for cancer: a pilot study.

PURPOSE: The objective of this study was to compare the effects of two types of enteral supplements, an antioxidant-enriched enteral nutrition (AeEN) and an immune-enhancing enteral nutrition (IeEN), on the nutrition, immunoinflammatory response, antioxidant capacity and clinical outcomes in patients after esophagectomy for cancer. METHODS: Patients (n = 20) undergoing esophagectomy for cancer were randomized in this single-center, open-label study. Two types of enteral supplements were used for 5 days before surgery and 7 days after surgery. The circulating levels of nutritional markers, immunoinflammatory markers, oxidative stress markers, and the antioxidant capacity were compared throughout the perioperative period, and the patients' clinical outcomes were also compared. RESULTS: The circulating levels of nutritional markers decreased after surgery, but the changes were not significantly different between the AeEN group and the IeEN group throughout the perioperative period. Surgery increased the immunoinflammatory markers, and the levels were not significantly different between the groups after surgery. Surgery also increased the levels of oxidative stress markers, but there were no significant differences between the groups throughout the study period. CONCLUSIONS: The results of this pilot study suggest that AeEN and IeEN have a similar effect on nutrition, the immunoinflammatory response, antioxidant capacity and clinical outcomes after esophagectomy for cancer. These findings, therefore, warrant further studies on a larger scale.

Esophagectomy for thoracic esophageal cancer with a double aortic arch: report of a case.

We report the case of a 50-year-old man with a double aortic arch who underwent esophagectomy for cancer in the middle thoracic esophagus at clinical Stage IIA (T3N0M0), based on the TNM classification (UICC 2002). The patient underwent esophagectomy with three-field lymphadenectomy following neoadjuvant chemotherapy. In such a case, it is important to recognize the anatomy in the upper mediastinum, especially the relationship between the right and left aortic arch, and the recurrent laryngeal nerves using computed tomography (CT) and three-dimensional CT. At first, we performed a cervical lymphadenectomy in order to isolate the bilateral recurrent laryngeal nerves, then mediastinal lymphadenectomy through a right thoracotomy. However, we could not confirm the bilateral recurrent laryngeal nerves during mediastinal lymphadenectomy, and were thus unable to resect them. The postoperative course was uneventful. The patient died of multiple liver metastasis 4 years after the surgery, with no evidence of recurrence in any lymph node.

Thermoresponsible cell adhesion/detachment on transparent nanocomposite films consisting of poly(2-methoxyethyl acrylate) and clay.

Soft, transparent and mechanically tough nanocomposite (M-NC) films composed of poly(2-methoxyethyl acrylate) (PMEA) and inorganic clay (hectorite) were studied as substrates for a living cell harvest system. It was found that five cell types could all be cultivated to confluence on the surface of M-NC n films with clay content (n = 10-23 wt%), although the cells hardly cultivated on the surface of chemically-cross-linked PMEA and linear PMEA films. Further, it was found that the cells cultured on the surfaces of M-NC films can be detached, without any enzymatic digestion, by decreasing the medium temperature and/or simultaneously using gentle pipetting. The detached cell was obtained as a single cell or a contiguous cell sheet, both of which were viable and recultured. From the compositions and surface properties, it was estimated that the cell culture and subsequent cell detachment were attributed to the unique PMEA/clay network. The new soft nanocomposite is potentially a very promising substrate for tissue engineering.

Photodynamic therapy for large superficial squamous cell carcinoma of the esophagus.

BACKGROUND: Photodynamic therapy (PDT) has been found to be safe and effective in patients with small early esophageal squamous cell carcinoma (SCC). However, its efficacy for widespread superficial SCC has not yet been confirmed. OBJECTIVE: To assess the long-term survival, complications, and recurrence of PDT for large superficial esophageal SCC. DESIGN: Retrospective study. SETTING: Tertiary referral center. PATIENTS: A total of 38 patients with superficial SCC of the esophagus. All patients had a large unifocal lesion or multifocal lesions that were too large to be resected endoscopically. In addition, all patients were physiologically unfit for esophagectomy or had refused surgery. INTERVENTIONS: PDT with porfimer sodium. MAIN OUTCOME MEASUREMENTS: Clinical follow-up, long-term survival, complications, and recurrence were evaluated. RESULTS: Thirty-one patients (82%) had mucosal cancer (T1m), and 7 (18%) had submucosal cancer (T1sm). No patient had lymph node involvement. Nineteen patients had other primary malignancies. Complete remission was achieved in 33 (87%). At the time of writing, 28 patients (74%) were alive without recurrence. After a median follow-up period of 64 months (range, 7-125 months) after PDT, the overall 5-year survival rate was 76%. There was no treatment-related mortality. LIMITATIONS: Retrospective study with a small number of patients. CONCLUSIONS: This long-term follow-up study revealed that PDT was a potentially curative treatment for large superficial esophageal SCC. PDT might be a reasonable alternative to esophagectomy or to endoscopic resection for patients with superficial SCC of the esophagus without lymph node metastasis.

Histone H3 lysine 4 methylation is associated with the transcriptional reprogramming efficiency of somatic nuclei by oocytes.

BACKGROUND: When the nuclei of mammalian somatic cells are transplanted to amphibian oocytes in the first meiotic prophase, they are rapidly induced to begin transcribing several pluripotency genes, including Sox2 and Oct4. The more differentiated the donor cells of the nuclei, the longer it takes for the pluripotency genes to be activated after the nuclear transfer to oocytes. We have used this effect in order to investigate the role of histone modifications in this example of nuclear reprogramming. RESULTS: Reverse transcription polymerase chain reaction analysis shows that the transcriptional reprogramming of pluripotency genes, such as Sox2 and Oct4, takes place in transplanted nuclei from C3H10T1/2 cells and from newly differentiated mouse embryonic stem cells. We find that the reprogramming of 10T1/2 nuclei is accompanied by an increased phosphorylation, an increased methylation and a rapidly reduced acetylation of several amino acids in H3 and other histones. These results are obtained by the immunofluorescent staining of transplanted nuclei and by Western blot analysis. We have also used chromatin immunoprecipitation analysis to define histone modifications associated with the regulatory or coding regions of pluripotency genes in transplanted nuclei. Histone phosphorylation is increased and histone acetylation is decreased in several regulatory and gene coding regions. An increase of histone H3 lysine 4 dimethylation (H3K4 me2) is seen in the regulatory regions and gene coding region of pluripotency genes in reprogrammed nuclei. Furthermore, histone H3 lysine 4 trimethylation (H3K4 me3) is observed more strongly in the regulatory regions of pluripotency genes in transplanted nuclei that are rapidly reprogrammed than in nuclei that are reprogrammed slowly and are not seen in beta-globin, a gene that is not reprogrammed. When 10T1/2 nuclei are incubated in Xenopus oocyte extracts, histone H3 serine 10 (H3S10) is strongly phosphorylated within a few hours. Immunodepletion of Aurora B prevents this phosphorylation. CONCLUSION: We conclude that H3K4 me2 and me3 are likely to be important for the efficient reprogramming of pluripotency genes in somatic nuclei by amphibian oocytes and that Aurora B kinase is required for H3S10 phosphorylation which is induced in transplanted somatic cell nuclei.

A p53-dependent checkpoint pathway prevents rereplication.

Eukaryotic cells control the initiation of DNA replication so that origins that have fired once in S phase do not fire a second time within the same cell cycle. Failure to exert this control leads to genetic instability. Here we investigate how rereplication is prevented in normal mammalian cells and how these mechanisms might be overcome during tumor progression. Overexpression of the replication initiation factors Cdt1 and Cdc6 along with cyclin A-cdk2 promotes rereplication in human cancer cells with inactive p53 but not in cells with functional p53. A subset of origins distributed throughout the genome refire within 2-4 hr of the first cycle of replication. Induction of rereplication activates p53 through the ATM/ATR/Chk2 DNA damage checkpoint pathways. p53 inhibits rereplication through the induction of the cdk2 inhibitor p21. Therefore, a p53-dependent checkpoint pathway is activated to suppress rereplication and promote genetic stability.

v-Crk activates the phosphoinositide 3-kinase/AKT pathway by utilizing focal adhesion kinase and H-Ras.

v-Crk, an oncogene product of avian sarcoma virus CT10, efficiently transforms chicken embryo fibroblasts (CEF). We have recently reported that constitutive activation of the phosphoinositide 3-kinase (PI3K)/AKT pathway plays a critical role in the v-Crk-induced transformation of CEF. In the present study we investigated the molecular mechanism by which v-Crk activates the PI3K/AKT pathway. First, we found that v-Crk promotes the association of the p85 regulatory subunit of PI3K with focal adhesion kinase (FAK) by inducing the phosphorylation of the Y397 residue in FAK. This FAK phosphorylation needs activation of the Src family tyrosine kinase(s) for which the v-Crk SH2 domain is responsible. v-Crk was unable to activate the PI3K/AKT pathway in FAK-null cells, indicating the functional importance of FAK. In addition, we found that H-Ras is also required for the activation of the PI3K/AKT pathway. The v-Crk-induced activation of AKT was greatly enhanced by the overexpression of H-Ras or its guanine nucleotide exchange factor mSOS, which binds to the v-Crk SH3 domain, whereas a dominant-negative mutant of H-Ras almost completely suppressed this activation. Furthermore, we showed that v-Crk stimulates the interaction of H-Ras with the Ras binding domain in the PI3K p110 catalytic subunit. Our data indicated that the v-Crk-induced activation of PI3K/AKT pathway was cooperatively achieved by two distinct interactions. One is the interaction of p85 with tyrosine-phosphorylated FAK promoted by the v-Crk SH2 domain, and another is the interaction of p110 with H-Ras dictated by the v-Crk SH3 domain.