Generation of Stratified Squamous Epithelial Progenitor Cells from Mouse Induced Pluripotent Stem Cells

Background: Application of induced pluripotent stem (iPS) cells in regenerative medicine will bypass ethical issues associated with use of embryonic stem cells. In addition, patient-specific IPS cells can be useful to elucidate the pathophysiology of genetic disorders, drug screening, and tailor-made medicine. However, in order to apply iPS cells to mitotic tissue, induction of tissue stem cells that give rise to progeny of the target organ is required. Methodology/Principal Findings: We induced stratified epithelial cells from mouse iPS cells by co-culture with PA6 feeder cells (SDIA-method) with use of BMP4. Clusters of cells positive for the differentiation markers KRT1 or KRT12 were observed in KRT14-positive colonies. We successfully cloned KRT14 and p63 double-positive stratified epithelial progenitor cells from iPS-derived epithelial cells, which formed stratified epithelial sheets consisting of five- to six-polarized epithelial cells in vitro. When these clonal cells were cultured on denuded mouse corneas, a robust stratified epithelial layer was observed with physiological cell polarity including high levels of E-cadherin, p63 and K15 expression in the basal layer and ZO-1 in the superficial layer, recapitulating the apico-basal polarity of the epithelium in vivo. Conclusions/Significance: These results suggest that KRT14 and p63 double-positive epithelial progenitor cells can b

Proton Spin Relaxation Induced by Quantum Tunneling in Fe8 Molecular Nanomagnet

The spin-lattice relaxation rate $T_{1}^{-1}$ and NMR spectra of $^1$H in single crystal molecular magnets of Fe8 have been measured down to 15 mK. The relaxation rate $T_1^{-1}$ shows a strong temperature dependence down to 400 mK. The relaxation is well explained in terms of the thermal transition of the iron state between the discreet energy levels of the total spin S=10. The relaxation time $T_1$ becomes temperature independent below 300 mK and is longer than 100 s. In this temperature region stepwise recovery of the $^1$H-NMR signal after saturation was observed depending on the return field of the sweep field. This phenomenon is attributed to resonant quantum tunneling at the fields where levels cross and is discussed in terms of the Landau-Zener transition.Comment: 13 pages, 5 figure

Eating-related distress and need for nutritional support of families of advanced cancer patients: A nationwide survey of bereaved family members

Background: A number of advanced cancer patients are suffering from physical and psychosocial burdens because of cancer cachexia, and these burdens also greatly impact on their family members and relationships between patients and family members. It is necessary to consider the psychosocial impact of cancer cachexia on family members of advanced cancer patients. Methods: A cross-sectional anonymous nationwide survey was conducted involving 925 bereaved family members of cancer patients who had been admitted to 133 inpatient hospices throughout Japan. Results: A total of 702 bereaved family members returned the questionnaires (response rate, 75.9%). Concerning eating-related distress, 'I served what the patient wanted without consideration of calories and nutritional composition' was highest (75.1%), and 'I tried making many kinds of meals for the patient' and 'I was concerned about planning meals for the patient every day' followed (63.0% and 59.4%, respectively). The top 5 of the 19 items were categorized as 'fighting back'. Need for nutritional support was high (72.2%), and need for explanations about the reasons for anorexia and weight loss of patients was moderate (41.4%). Explanatory factor analysis of eating-related distress identified the following four domains: (factor 1) feeling that family members forced the patient to eat to avoid death, (factor 2) feeling that family members made great efforts to help the patient eat, (factor 3) feeling that eating was a cause of conflicts between the patient and family members, and (factor 4) feeling that correct information was insufficient. Results of multiple logistic regression analysis showed that spouse, fair/poor mental status, factors 1, and 4 were identified as independent determinants of major depression (odds ratio [OR] 3.27 [95% confidence interval (CI) 1.24-8.60], P=0.02; OR 4.50 [95% CI 2.46-8.25], P<0.001; OR 2.51 [95% CI 1.16-5.45], P=0.02; OR 2.33 [95% CI 1.13-4.80], P=0.02, respectively). Conclusions: A number of family members of advanced cancer patients experienced high levels of eating-related distress and had a need for nutritional support

Trust in Physicians, Continuity and Coordination of Care, and Quality of Death in Patients with Advanced Cancer

Background: Provider-centered factors contribute to unexplained variation in the quality of death (QOD). The relationship between healthcare providers (HCPs) and patients, bidirectional communication, and consistency of longitudinal care planning are important provider-centered factors.Objective: To explore whether the level of trust in HCPs, the quality of continuity of care, and the level of coordination of care among home HCPs are associated with the QOD for cancer patients dying at home.Design: This study was a part of a nationwide multicenter questionnaire survey of bereaved family members of cancer patients evaluating the quality of end-of-life care in Japan.Setting/Subjects: We investigated 702 family members of cancer patients who died at home.Measurements: The QOD was evaluated from nine core domains of the short version of the Good Death Inventory (GDI). We measured five factors on a Likert scale, including patient and family trust in HCPs, continuity of care by home hospice and hospital physicians, and coordination of care among home hospice staff.Results: A total of 538 responses (77%) were obtained and 486 responses were analyzed. Trust in HCPs was correlated with the GDI score (r = 0.300–0.387, p < 0.001). The quality of care coordination was associated with the GDI score (r = 0.242, p < 0.001).Conclusions: Trust of the patient and family in home hospice staff, as well as coordination of care among hospice staff, are associated with the QOD for cancer patients dying at home

Transcriptionally linked simultaneous overexpression of P450 genes for broad-spectrum herbicide resistance

雑草が獲得した最強の除草剤抵抗性メカニズムの解明 --解毒酵素の一斉活性化--. 京都大学プレスリリース. 2023-06-14.Broad-spectrum herbicide resistance (BSHR), often linked to weeds with metabolism-based herbicide resistance, poses a threat to food production. Past studies have revealed that overexpression of catalytically promiscuous enzymes explains BSHR in some weeds; however, the mechanism of BSHR expression remains poorly understood. Here, we investigated the molecular basis of high-level resistance to diclofop-methyl in BSHR late watergrass (Echinochloa phyllopogon) found in the United States, which cannot be solely explained by the overexpression of promiscuous cytochrome P450 monooxygenases CYP81A12/21. The BSHR late watergrass line rapidly produced 2 distinct hydroxylated diclofop acids, only 1 of which was the major metabolite produced by CYP81A12/21. RNA-seq and subsequent reverse transcription quantitative PCR (RT-qPCR)-based segregation screening identified the transcriptionally linked overexpression of a gene, CYP709C69, with CYP81A12/21 in the BSHR line. The gene conferred diclofop-methyl resistance in plants and produced another hydroxylated diclofop acid in yeast (Saccharomyces cerevisiae). Unlike CYP81A12/21, CYP709C69 showed no other herbicide-metabolizing function except for a presumed clomazone-activating function. The overexpression of the 3 herbicide-metabolizing genes was also identified in another BSHR late watergrass in Japan, suggesting a convergence of BSHR evolution at the molecular level. Synteny analysis of the P450 genes implied that they are located at mutually independent loci, which supports the idea that a single trans-element regulates the 3 genes. We propose that transcriptionally linked simultaneous overexpression of herbicide-metabolizing genes enhances and broadens the metabolic resistance in weeds. The convergence of the complex mechanism in BSHR late watergrass from 2 countries suggests that BSHR evolved through co-opting a conserved gene regulatory system in late watergrass

Serum amyloid A-induced IL-6 production by rheumatoid synoviocytes

AbstractIn this study, we investigated the role of serum amyloid A protein (SAA) in the production of interleukin-6 (IL-6) using rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). Recombinant SAA stimulation induced the production of pro-inflammatory cytokine, IL-6, from RA-FLS. The signaling events induced by SAA included the activation of the mitogen-activated protein kineases, p38 and JNK1/2 and the activation of nuclear factor-kappa B (NF-κB). Inhibitor studies have shown SAA-induced IL-6 production to be down-regulated by NF-κB inhibition and partially inhibited by p38 or JNK inhibitors. Our findings demonstrate that SAA is a significant inducer of IL-6, which is critically involved in RA pathogenesis

Deregulated GSK3β sustains gastrointestinal cancer cells survival by modulating human telomerase reverse transcriptase and telomerase

金沢大学がん研究所分子標的がん医療研究開発センターPurpose: Glycogen synthase kinase-3β (GSK3β) regulates multiple cell signaling pathways and has been implicated in glucose intolerance, neurodegenerative disorders, and inflammation. We investigated the expression, activity, and putative pathologic role of GSK3β in gastrointestinal, pancreatic, and liver cancers. Experimental Design: Colon, stomach, pancreatic, and liver cancer cell lines; nonneoplastic HEK293 cells; and matched pairs of normal and tumor tissues of stomach and colon cancer patients were examined for GSK3β expression and its phosphorylation at serine 9 (inactive form) and tyrosine 216 (active form) by Western immunoblotting and for GSK3β activity by in vitro kinase assay. The effects of small-molecule GSK3β inhibitors and of RNA interference on cell survival, proliferation, and apoptosis were examined in vitro and on human colon cancer cell xenografts in athymic mice. The effects of GSK3β inhibition on human telomerase reverse transcriptase (hTERT) expression and telomerase activity were compared between colon cancer and HEK293 cells. Results: Cancer cell lines and most cancer tissues showed increased GSK3β expression and increased tyrosine 216 phosphorylation and activity but decreased serine 9 phosphorylation compared with HEK293 cells and nonneoplastic tissues. Inhibition of GSK3β resulted in attenuated cell survival and proliferation and increased apoptosis in most cancer cell lines and in HT-29 xenografts in rodents but not in HEK293 cells. GSK3β inhibition in colon cancer cells was associated with decreased hTERT expression and telomerase activity. Conclusion: The results indicate that deregulated GSK3β sustains gastrointestinal cancer cells survival through modulation of hTERT and telomerase. © 2009 American Association for Cancer Research

Serum amyloid A triggers the mosodium urate -mediated mature interleukin-1β production from human synovial fibroblasts

Background: Monosodium urate (MSU) has been shown to promote inflammasome activation and interleukin-1β (IL-1β) secretion in monocyte/macrophages, but the cellular pathway and nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation in synovial tissues, remain elusive. In this study, we investigated the effects of MSU on synovial fibroblasts to elucidate the process of MSU-mediated synovial inflammation.Methods: Human synovial fibroblasts were stimulated with MSU in the presence or absence of serum amyloid A (SAA). The cellular supernatants were analyzed by immunoblotting using anti-IL-1β or anti-caspase-1 antibodies. IL-1β or NLRP3 mRNA expressions were analyzed by real-time PCR or reverse transcription-PCR (RT-PCR) method.Results: Neither SAA nor MSU stimulation resulted in IL-1β or interleukin-1α (IL-1α) secretions and pro-IL-1β processing in synovial fibroblasts. However, in SAA-primed synovial fibroblasts, MSU stimulation resulted in the activation of caspase-1 and production of active IL-1β and IL-1α. The effect of SAA on IL-1β induction was impaired in cells by silencing NLRP3 using siRNA or treating with caspase-1 inhibitor. In addition, SAA induced the secretion of cathepsin B and NLRP3 mRNA expression in synovial fibroblasts.Conclusions: Our data demonstrate that exposure of human synovial fibroblasts to SAA promotes MSU-mediated caspase-1 activation and IL-1β secretion in the absence of microbial stimulation. These findings provide insight into the molecular processes underlying the synovial inflammatory condition of gout