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Objectives A small subpopulation of colorectal cancer stem cells (CSCs) possess the ability to self-renew and the capacity to initiate the original tumor. EpCAMhigh/CD44+ cells are regarded as CSCs in colorectal cancer. The present study was undertaken to investigate the significance of EpCAM in the in vitro proliferation ability and oxaliplatin chemoresistance of EpCAMhigh/CD44+ colorectal CSCs. Methods We applied fluorescence-activated cell sorting (FACS) to separate the EpCAMhigh/CD44+ subset from human colorectal cancer cell line HCT116. We also used siRNA targeting EpCAM to create EpCAM−/CD44+ subpopulation. Then we compared EpCAMhigh/CD44+ cells and EpCAM−/CD44+ cells for proliferation ability and the chemoresistance to oxaliplatin by CCK8 assay. Results The EpCAMhigh/CD44+ subset comprises almost 6.25 ± 0.09% in cell line HCT116, and the EpCAM−/CD44+ cells displayed a significantly lower proliferation ability and weaker oxaliplatin chemoresistance than the EpCAMhigh/CD44+ cells. Conclusions EpCAM is critical for tumor proliferation and oxaliplatin chemoresistance in EpCAMhigh/CD44+ colorectal CSCs.

Following tissue injury, latent sensitization (LS) of nociceptive signaling can persist indefinitely, kept in remission by compensatory µ-opioid receptor constitutive activity (MOR(CA)) in the dorsal horn of the spinal cord. To demonstrate LS, we conducted plantar incision in mice and then waited 3–4 weeks for hypersensitivity to resolve. At this time (remission), systemic administration of the opioid receptor antagonist/inverse agonist naltrexone reinstated mechanical and heat hypersensitivity. We first tested the hypothesis that LS extends to serotonergic neurons in the rostral ventral medulla (RVM) that convey pronociceptive input to the spinal cord. We report that in male and female mice, hypersensitivity was accompanied by increased Fos expression in serotonergic neurons of the RVM, abolished on chemogenetic inhibition of RVM 5-HT neurons, and blocked by intrathecal injection of the 5-HT(3)R antagonist ondansetron; the 5-HT(2A)R antagonist MDL-11 939 had no effect. Second, to test for MOR(CA), we microinjected the MOR inverse agonist d-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) and/or neutral opioid receptor antagonist 6β-naltrexol. Intra-RVM CTAP produced mechanical hypersensitivity at both hindpaws; 6β-naltrexol had no effect by itself, but blocked CTAP-induced hypersensitivity. This indicates that MOR(CA), rather than an opioid ligand-dependent mechanism, maintains LS in remission. We conclude that incision establishes LS in descending RVM 5-HT neurons that drives pronociceptive 5-HT(3)R signaling in the dorsal horn, and this LS is tonically opposed by MOR(CA) in the RVM. The 5-HT(3) receptor is a promising therapeutic target for the development of drugs to prevent the transition from acute to chronic postsurgical pain. SIGNIFICANCE STATEMENT Surgery leads to latent pain sensitization and a compensatory state of endogenous pain control that is maintained long after tissue healing. Here, we show that either chemogenetic inhibition of serotonergic neuron activity in the RVM or pharmacological inhibition of 5-HT(3) receptor signaling at the spinal cord blocks behavioral signs of postsurgical latent sensitization. We conclude that MOR(CA) in the RVM opposes descending serotonergic facilitation of LS and that the 5-HT(3) receptor is a promising therapeutic target for the development of drugs to prevent the transition from acute to chronic postsurgical pain.

Aims: The present study investigated the exact proportion, the extent of in vitro proliferation potential, and oxaliplatin chemoresistance of EpCAMhigh/CD44+ cancer stem cells in colorectal cancer. Its underlying mechanism was also explored. Background: Colorectal cancer stem cells (CSC) play crucial roles in tumorigenicity and chemoresistance. Multiple studies have shown that JAK/STAT, NOTCH, and Wnt/-catenin pathways, associated with tumour recurrence and metastasis, contribute to the proliferation and maintenance of CSCs. CSCs become resistant to chemo-radiotherapies by improving DNA damage repair, changing cell cycle checkpoints, and scavenging reactive oxygen species, resulting in a bad patient prognosis. Objective: This work was carried out to determine the precise fraction, the degree of in vitro proliferation capability, and the level of oxaliplatin chemoresistance exhibited by EpCAMhigh/CD44+ cancer stem cells in colorectal cancer. The research was also done to investigate its underlying process. Methods: Fluorescence-activated cell sorting (FACS) was applied to isolate the EpCAMhigh/CD44+ populations from three human colorectal cancer cell lines (HCT116, HT29, and LoVo), and we quantified the average proportion of the EpCAMhigh/CD44+ cells in every cell lines. The comparison of their proliferation ability and the chemoresistance to oxaliplatin with the parental cells was estimated by CCK8 assay. The activated signaling pathway was tested by Western Blotting. result: EpCAMhigh/CD44+ subpopulation comprise about 4.98±1.24% of the total human colorectal cancer cell lines, and the EpCAMhigh/CD44+ cells exhibited a highly better proliferation ability and stronger oxaliplatin chemoresistance than the parental cells. Wnt/β-catenin signaling pathway is activated in EpCAMhigh/CD44+ HCT116 cells. Results: EpCAMhigh/CD44+ subpopulation comprises about 4.98±1.24% of the total human colorectal cancer cell lines, and the EpCAMhigh/CD44+ cells exhibited a highly better proliferation ability and stronger oxaliplatin chemoresistance than the parental cells. The wnt/β-catenin signaling pathway is activated in EpCAMhigh/CD44+ HCT116 cells conclusion: Activation of Wnt/β-Catenin signaling in EpCAMhigh/CD44+ cells endow colorectal cancer with tumor proliferation and oxaliplatin chemoresistance. Conclusion: Activation of Wnt/β-Catenin signaling in EpCAMhigh/CD44+ cells endow colorectal cancer with tumor proliferation and oxaliplatin chemoresistance.

Peripheral nerve injuries result in pronounced alterations in dorsal root ganglia, which can lead to the development of neuropathic pain. Although the polymodal mechanosensitive transient receptor potential ankyrin 1 (TRPA1) ion channel is emerging as a relevant target for potential analgesic therapies, preclinical studies do not provide unequivocal mechanistic insight into its relevance for neuropathic pain pathogenesis. By using a transgenic mouse model with a conditional depletion of the interleukin-6 (IL-6) signal transducer gp130 in Nav1.8 expressing neurons (SNS-gp130-/-), we provide a mechanistic regulatory link between IL-6/gp130 and TRPA1 in the spared nerve injury (SNI) model. Spared nerve injury mice developed profound mechanical hypersensitivity as indicated by decreased withdrawal thresholds in the von Frey behavioral test in vivo, as well as a significant increase in mechanosensitivity of unmyelinated nociceptive primary afferents in ex vivo skin-nerve recordings. In contrast to wild type and control gp130fl/fl animals, SNS-gp130-/- mice did not develop mechanical hypersensitivity after SNI and exhibited low levels of Trpa1 mRNA in sensory neurons, which were partially restored by adenoviral gp130 re-expression in vitro. Importantly, uninjured but not injured neurons developed increased responsiveness to the TRPA1 agonist cinnamaldehyde, and neurons derived from SNS-gp130-/- mice after SNI were significantly less responsive to cinnamaldehyde. Our study shows for the first time that TRPA1 upregulation is attributed specifically to uninjured neurons in the SNI model, and this depended on the IL-6 signal transducer gp130. We provide a solution to the enigma of TRPA1 regulation after nerve injury and stress its significance as an important target for neuropathic pain disorders.

Introduction: Overexpression of cellular repressor of E1A-stimulated genes 1 (CREG1) promotes the formation of compact, contracting myocardium-like structures during cardiomyogenesis from embryonic stem cells. Cell biology studies suggest that CREG1 promotes differentiation and inhibits proliferation of developing cardiomyocytes. This study aims to understand the role of CREG1 during myocardial development in vivo . Methods: Cardiac tissues were harvested from wild-type (WT) and constitutive Creg1 knockout (KO) postnatal day 1.5 and 17 through timed mating of heterozygous Creg1 mice. Creg1 KO mice were compared with Creg1 WT littermates using immunoblotting, immunofluorescence, and methylene blue staining. Results: Postnatal day 1.5 Creg1 KO hearts showed lower levels of expression of slow skeletal muscle troponin I (TnI) compared with WT counterparts. No significant difference in expression was seen between WT and KO Creg1 hearts for myosin light chain 3 (MLC3), myosin heavy chain (MHC), and cardiac troponin I. Immunofluorescence staining revealed increased numbers of PCNA (Proliferating Cell Nuclear Antigen) positive nuclei in postnatal day 1.5 Creg1 KO hearts, suggesting enhanced cardiomyocyte proliferation after loss of CREG1. Methylene blue staining in postnatal day 17 hearts displayed decreased myocardial compaction in Creg1 KO mice. Conclusion: CREG1 may promote cardiomyocyte differentiation and inhibit proliferation during postnatal myocardial development. Ablation of CREG1 results in cardiac compaction defects. Our findings suggest an important role for CREG1 in cardiac development.

CREG1 is a small glycoprotein which has been proposed as a transcription repressor, a secretory ligand, a lysosomal, or a mitochondrial protein. This is largely because of lack of antibodies for immunolocalization validated through gain- and loss-of-function studies. In the present study, we demonstrate, using antibodies validated for immunofluorescence microscopy, that CREG1 is mainly localized to the endosomal-lysosomal compartment. Gain- and loss-of-function analyses reveal an important role for CREG1 in both macropinocytosis and clathrin-dependent endocytosis. CREG1 also promotes acidification of the endosomal-lysosomal compartment and increases lysosomal biogenesis. Functionally, overexpression of CREG1 enhances macroautophagy/autophagy and lysosome-mediated degradation, whereas knockdown or knockout of CREG1 has opposite effects. The function of CREG1 in lysosomal biogenesis is likely attributable to enhanced endocytic trafficking. Our results demonstrate that CREG1 is an endosomal-lysosomal protein implicated in endocytic trafficking and lysosomal biogenesis. Abbreviations: AIFM1/AIF: apoptosis inducing factor mitochondria associated 1; AO: acridine orange; ATP6V1H: ATPase H+ transporting V1 subunit H; CALR: calreticulin; CREG: cellular repressor of E1A stimulated genes; CTSC: cathepsin C; CTSD: cathepsin D; EBAG9/RCAS1: estrogen receptor binding site associated antigen 9; EIPA: 5-(N-ethyl-N-isopropyl)amiloride; ER: endoplasmic reticulum; GFP: green fluorescent protein; HEXA: hexosaminidase subunit alpha; IGF2R: insulin like growth factor 2 receptor; LAMP1: lysosomal associated membrane protein 1; M6PR: mannose-6-phosphate receptor, cation dependent; MAPK1/ERK2: mitogen-activated protein kinase 1; MTORC1: mechanistic target of rapamycin kinase complex 1; PDIA2: protein disulfide isomerase family A member 2; SQSTM1/p62: sequestosome 1; TF: transferrin; TFEB: transcription factor EB

The cultivation of humanistic quality of resident doctors plays an important role in the development of individual physicians and the effective operation of the medical industry. Competence-oriented humanistic literacy training for resident and training doctors needs to integrate humanistic literacy into professional training to solve the existing problems in humanistic quality of current resident and training doctors. This paper mainly analyzes the necessity and shortcomings of the competency oriented humanistic quality training of resident and training doctors, and puts forward some measures to improve the humanistic quality of resident and training doctors.

Objective: To explore the clinical effect of domestic capsosol foam sclerotherapy in the treatment of hemorrhoids under transparent cap assisted endoscope. Methods: According to the experimental requirements, 120 cases of hemorrhoids hospitalized in gastroenterology department in our hospital from February to December in 2019 were selected as the treatment group. The foam sclerotherapy of polidocanol and the air in accordance with the 1:4 ratio was injected into varicose hemorrhoids and hemorrhoids through a transparent cap assisted endoscope. At the same time, 100 patients were selected as the control group and the patients were treated according to the routine protocol. The symptom score and clinical efficacy of the two groups were observed under different treatment schemes. Results: 120 patients were successfully injected with foam sclerotherapy under endoscope. There were significant differences in clinical symptom score and clinical efficacy between the injection sclerotherapy group and the control group. There were statistically significant differences in the bleeding, prolapse, painful defecation, anal foreign body sensation, impact on daily work, and hemorrhoids data between the treatment group and the control group (P

Glutamine (Gln) is a non-essential amino acid central for generating building blocks and cellular energy in tumours and rapidly proliferating non-transformed cells. However, the influence of Gln on regulating chromosomal stability of transformed and non-transformed cells remain poorly understand. We hypothesised that Gln is required for maintaining a homeostatic level of chromosomal stability. To this end, transformed cells HeLa and A375 and non-transformed cells NCM460 and HUVEC cells were intervened with varying concentrations of Gln (10, 1, 0.1 and 0.01 mM), with or without cisplatin (0.1 µg/ml), for 24 h. The cytokinesis-block micronucleus (MN) assay was used to determine chromosomal instability (CIN), the extent of which is reflected by the frequency of MN, nucleoplasmic bridge (NPB) and nuclear bud (NB). We demonstrated an unexpected decrease in the spontaneous rate of MN, but not NPB and NB, after Gln restriction in HeLa and A375 cells. Gln restriction reduced cisplatin-induced MN, but not NPB and NB, in HeLa and A375 cells. We further revealed that Gln restriction suppressed the proliferation of HeLa cells with high CIN induced by nocodazole, partially explaining why Gln restriction decreased the frequency of spontaneous and cisplatin-induced MN in transformed cells. In contrast, Gln restriction increased MN and NB, but not NPB, in NCM460 cells. In HUVEC cells, Gln restriction increased MN, NPB and NB. Meanwhile, Gln restriction sensitised NCM460 cells to cisplatin-induced genotoxicity. A similar but more pronounced pattern was observed in HUVEC cells. Collectively, these results suggest that the in vitro influences of Gln metabolism on CIN depend on cellular contexts: Transformed cells require high Gln to fine tune their CIN in an optimal rate to maximise genomic heterogeneity and fitness, whereas non-transformed cells need high Gln to prevent CIN.

Elevated Homocysteine (Hcy) is associated with increased risk of vascular disease, but whether it induces genotoxicity to vascular endothelial cells remains unknown. Here, we conducted a comprehensive study of the genotoxicity, and unexpected anti-genotoxicity, of Hcy by cytokinesis-blocked micronucleus assay in HUVECs and erythrocyte micronucleus test in mouse bone marrow cells. Our experiments led to several important findings. First, while supraphysiological Hcy (SP-Hcy) exhibited remarkable genotoxicity, physiologically-relevant Hcy (PR-Hcy) reduced the basal genotoxicity. Second, among the metabolites of Hcy, cysteine phenocopied the anti-genotoxicity of PR-Hcy and, methionine, S-adenosylhomocysteine and H2S phenocopied the genotoxicity of SP-Hcy. Third, the genotoxicity of SP-Hcy was mitigated by vitamin B6, Fe2+ and Cu2+, but was exacerbated by N-acetylcysteine. Fourth, under pre-, co- or post-treatment protocol, both SP-Hcy and PR-Hcy attenuated the genotoxicity of cisplatin, mitomycin-C, nocodazole or deoxycholate. Finally, 100 and 250 mg/kg Hcy ameliorated cisplatin-induced genotoxicity in bone marrow cells of CF-1 and Kunming mice. Our results suggest that genotoxicity may be one mechanism through which Hcy confers an increased risk for vascular disease, but more importantly, they challenge the long-standing paradigm that Hcy is always harmful to human health. Our study calls for a more systematic effort in understanding the molecular mechanisms underlying the anti-genotoxicity of Hcy.

Gravity plays an important role in normal tissue maintenance. The ability of stem cells to repair tissue loss in space through regeneration and differentiation remains largely unknown. To investigate the impact of microgravity on blood vessel formation from pluripotent stem cells, we employed the embryoid body (EB) model for vasculogenesis and simulated microgravity by clinorotation. We first differentiated mouse embryonic stem cells into cystic EBs containing two germ layers and then analyzed vessel formation under clinorotation. We observed that endothelial cell differentiation was slightly reduced under clinorotation, whereas vascular branch morphogenesis was markedly enhanced. EB-derived endothelial cells migrated faster, displayed multiple cellular processes, and had higher Cdc42 and Rac1 activity when subjected to clinorotation. Genetic analysis and rescue experiments demonstrated that Cdc42 but not Rac1 is required for microgravity-induced vascular branch morphogenesis. Furthermore, affinity pull-down assay and mass spectrometry identified Rap1GDS1 to be a Cdc42 guanine nucleotide exchange factor, which was upregulated by clinorotation. shRNA-mediated knockdown of Rap1GDS1 selectively suppressed Cdc42 activation and inhibited both baseline and microgravity-induced vasculogenesis. This was rescued by ectopic expression of constitutively active Cdc42. Taken together, these results support the notion that simulated microgravity activates Cdc42 via Rap1GDS1 to promote vascular branch morphogenesis.

The tumor suppressor PTEN is essential for epithelial morphogenesis. Qi et al. identify Abi1, a core adaptor protein in the WAVE regulatory complex, as a new PTEN substrate. PTEN dephosphorylates Abi1 and causes Abi1 degradation through calpains and thus down-regulates the WAVE regulatory complex to induce epithelial differentiation and polarization., The tumor suppressor PTEN is essential for early development. Its lipid phosphatase activity converts PIP3 to PIP2 and antagonizes the PI3K–Akt pathway. In this study, we demonstrate that PTEN’s protein phosphatase activity is required for epiblast epithelial differentiation and polarization. This is accomplished by reconstitution of PTEN-null embryoid bodies with PTEN mutants that lack only PTEN’s lipid phosphatase activity or both PTEN’s lipid and protein phosphatase activities. Phosphotyrosine antibody immunoprecipitation and mass spectrometry were used to identify Abi1, a core component of the WASP-family verprolin homologous protein (WAVE) regulatory complex (WRC), as a new PTEN substrate. We demonstrate that PTEN dephosphorylation of Abi1 at Y213 and S216 results in Abi1 degradation through the calpain pathway. This leads to down-regulation of the WRC and reorganization of the actin cytoskeleton. The latter is critical to the transformation of nonpolar pluripotent stem cells into the polarized epiblast epithelium. Our findings establish a link between PTEN and WAVE-Arp2/3–regulated actin cytoskeletal dynamics in epithelial morphogenesis.

Understanding the regulation of cell-cell interactions during the formation of compact myocardial structures is important for achieving true cardiac regeneration through enhancing the integration of stem cell-derived cardiomyocytes into the recipient myocardium. In this study, we found that cellular repressor of E1A-stimulated genes 1 (CREG1) is highly expressed in both embryonic and adult hearts. Gain- and loss-of-function analyses demonstrated that CREG1 is required for differentiation of mouse embryonic stem (ES) cell into cardiomyocytes and the formation of cohesive myocardium-like structures in a cell-autonomous fashion. Furthermore, CREG1 directly interacts with Sec8 of the exocyst complex, which tethers vesicles to the plasma membrane. Site-directed mutagenesis and rescue of CREG1 knockout ES cells showed that CREG1 binding to Sec8 is required for cardiomyocyte differentiation and cohesion. Mechanistically, CREG1, Sec8, and N-cadherin colocalize at intercalated discs in vivo and are enriched at cell-cell junctions in cultured cardiomyocytes. CREG1 overexpression enhances the assembly of adherens and gap junctions. By contrast, its knockout inhibits the Sec8-N-cadherin interaction and induces their degradation. These results suggest that the CREG1 binding to Sec8 enhances the assembly of intercellular junctions and promotes cardiomyogenesis.

TNF-α-converting enzyme, a member of the ADAM (A disintegrin and metalloproteinase) protease family and also known as ADAM17, regulates inflammation and regeneration in health and disease. ADAM17 targets are involved in pain development and hypersensitivity in animal models of inflammatory and neuropathic pain. However, the role of ADAM17 in the pain pathway is largely unknown. Therefore, we used the hypomorphic ADAM17 (ADAM17ex/ex) mouse model to investigate the importance of ADAM17 in nociceptive behavior, morphology, and function of primary afferent nociceptors. ADAM17ex/ex mice were hyposensitive to noxious stimulation, showing elevated mechanical thresholds as well as impaired heat and cold sensitivity. Despite these differences, skin thickness and innervation were comparable to controls. Although dorsal root ganglia of ADAM17ex/ex mice exhibited normal morphology of peptidergic and nonpeptidergic neurons, a small but significant reduction in the number of isolectin β-4–positive neurons was observed. Functional electrical properties of unmyelinated nociceptors showed differences in resting membrane potential, afterhyperpolarization, and firing patterns in specific subpopulations of sensory neurons in ADAM17ex/ex mice. However, spinal cord morphology and microglia activity in ADAM17ex/ex mice were not altered. Our data suggest that ADAM17 contributes to the processing of painful stimuli, with a complex mode of action orchestrating the function of neurons along the pain pathway.—Quarta, S., Mitrić, M., Kalpachidou, T., Mair, N., Schiefermeier-Mach, N., Andratsch, M., Qi, Y., Langeslag, M., Malsch, P., Rose-John, S., Kress, M. Impaired mechanical, heat, and cold nociception in a murine model of genetic TACE/ADAM17 knockdown.

Yanmei Qi,1 Fengqiang Zhou,2 Lu Zhang,2 Lei Liu,2 Hong Xu,2 Huiguang Guo2 1Department of Gastroenterology, 2Department of General Surgery, Binzhou People’s Hospital, Binzhou, Shandong, People’s Republic of China Background: Prior study indicates that abnormal protein expression and functional changes in the development and progression of colorectal cancer is related to gene expression. The aim of this study was to construct an interference plasmid targeting the Ep-CAM gene and to investigate its effects on the proliferation of colorectal cancer cells. Methods: In this study, HT-29 and HCT-116 colorectal cancer cell lines were selected as cell models. The double-stranded micro (mi)RNA oligo was inserted into the pcDNATM6.2-GW/EmGFPmiR vector, which is an expression of miRNA. Lipofectamine™ 2000 was used to transfer plasmid into the empty plasmid group (transfected pcDNATM6.2-GW/EmGFPmiR-neg) and the interference group (transfected pcDNATM6.2-GW/EmGFPmiR-Ep-CAM-1), respectively. Meanwhile, the nontransferred HT-29 and HCT-116 acts as the blank control group. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the transfection efficiency. Western blot was used to detect Ep-CAM protein expression. The cell proliferation in each group was detected by using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results:The results indicated that the Ep-CAM messenger (m)RNA expression in the interference group was lower significantly compared with that of the empty plasmid group and control group (P

At its most fundamental level, touch sensation requires the translation of mechanical energy into mechanosensitive ion channel opening, thereby generating electro-chemical signals. Our understanding of this process, especially how the cytoskeleton influences it, remains unknown. Here we demonstrate that mice lacking the α-tubulin acetyltransferase Atat1 in sensory neurons display profound deficits in their ability to detect mechanical stimuli. We show that all cutaneous afferent subtypes, including nociceptors have strongly reduced mechanosensitivity upon Atat1 deletion, and that consequently, mice are largely insensitive to mechanical touch and pain. We establish that this broad loss of mechanosensitivity is dependent upon the acetyltransferase activity of Atat1, which when absent leads to a decrease in cellular elasticity. By mimicking α-tubulin acetylation genetically, we show both cellular rigidity and mechanosensitivity can be restored in Atat1 deficient sensory neurons. Hence, our results indicate that by influencing cellular stiffness, α-tubulin acetylation sets the force required for touch. DOI: http://dx.doi.org/10.7554/eLife.20813.001

Cellular repressor of E1A-stimulated genes 1 (CREG1) is a 24 kD glycoprotein essential for early embryonic development. Our immunofluorescence studies revealed that CREG1 is highly expressed at myocyte junctions in both embryonic and adult hearts. To explore it role in cardiomyogenesis, we employed gain- and loss-of-function analyses demonstrating that CREG1 is required for the differentiation of mouse embryonic stem (ES) cell into cohesive myocardium-like structures. Chimeric cultures of wild-type and CREG1 knockout ES cells expressing cardiac-specific reporters showed that the cardiomyogenic effect of CREG1 is cell autonomous. Furthermore, we identified a novel interaction between CREG1 and Sec8 of the exocyst complex, which tethers vesicles to the plasma membrane. Mutations of the amino acid residues D141 and P142 to alanine in CREG1 abolished its binding to Sec8. To address the role of the CREG1-Sec8 interaction in cardiomyogenesis, we rescued CREG1 knockout ES cells with wild-type and Sec8-binding mutant CREG1 and showed that CREG1 binding to Sec8 promotes cardiomyocyte differentiation and cohesion. Mechanistically, CREG1, Sec8 and N-cadherin all localize at cell-cell adhesion sites. CREG1 overexpression enhances the assembly of adherens and gap junctions. By contrast, its knockout inhibits the Sec8-N-cadherin interaction and induces their degradation. Finally, shRNA-mediated knockdown of Sec8 leads to cardiomyogenic defects similar to CREG1 knockout. These results suggest that the CREG1 binding to Sec8 enhances the assembly of intercellular junctions and promotes cardiomyogenesis.

During early embryogenesis, endodermal γ1-laminin expression is required for basement membrane (BM) assembly, promoting conversion of non-polar pluripotent cells into polarized epiblast. The influence of laminin-111 (Lm111) and its integrin and dystroglycan (DG) receptors on epiblast in embryoid bodies (EBs), a model for differentiation of the embryonic plate, was further investigated. Lm111 added to the medium of EBs initiated conversion of inner nonpolar cell to the polarized epiblast epithelium with an exterior-to-central basal-to-apical orientation. Microinjection of Lm111 into EB interiors resulted in an interior BM with complete inversion of cell polarity. Lm111 assembled a BM on integrin-β1 null EBs with induction of polarization at reduced efficiency. β-Integrin compensation was not detected in these nulls with integrin adaptor proteins failing to assemble. A dimer of laminin LG domains 4–5 (LZE3) engineered to strongly bind to α-dystroglycan almost completely inhibited laminin accumulation on integrin β1-null EBs, reducing BM and ablating cell polarization. When Lm111 was incubated with integrin-β1/dystroglycan double-knockout EBs, laminin failed to accumulate on the EBs, the EBs did not differentiate, and the EBs underwent apoptosis. Collectively the findings support the hypotheses that the locus of laminin cell surface assembly can determine the axis of epithelial polarity. This requires integrin- and/ or dystroglycan-dependent binding to laminin LG domains with the highest efficiency achieved when both receptors are present. Finally, EBs that cannot assemble a matrix undergo apoptosis.

Apoptosis during epithelial lumen formation is mediated by hypoxia-induced expression of the proapoptotic protein Bnip3, which is promoted by AIF-mediated reactive oxygen species production and HIF-2α stabilization., Apoptosis is an essential step in cavitation during embryonic epithelial morphogenesis, but its mechanisms are largely unknown. In this paper, we used embryonic stem cell–differentiated embryoid bodies (EBs) as a model and found that Bnip3 (Bcl-2/adenovirus E1B 19-kD interacting protein), a BH3-only proapoptotic protein, was highly up-regulated during cavitation in a hypoxia-dependent manner. Short hairpin RNA silencing of Bnip3 inhibited apoptosis of the core cells and delayed cavitation. We show that the Bnip3 up-regulation was mediated mainly by hypoxia-inducible factor (HIF)–2. Ablation of HIF-2α or HIF-1β, the common β subunit of HIF-1 and -2, suppressed Bnip3 up-regulation and inhibited apoptosis and cavitation. We further show that apoptosis-inducing factor (AIF) cooperated with Bnip3 to promote lumen clearance. Bnip3 silencing in AIF-null EBs nearly blocked apoptosis and cavitation. Moreover, AIF also regulated Bnip3 expression through mitochondrial production of reactive oxygen species and consequent HIF-2α stabilization. These results uncover a mechanism of cavitation through hypoxia-induced apoptosis of the core cells mediated by HIFs, Bnip3, and AIF.

Genetic inactivation of the transcription factor GATA-6 in the embryoid body induces massive apoptosis at the early stage of ES cell differentiation. Evidence is provided that BMP-2 is a direct transcription target of GATA-6 and mediates GATA-6-dependent cell survival in concert with endoderm-derived basement membrane., GATA-6 is a zinc-finger transcription factor essential for early embryogenesis. Ablation of GATA-6 in mice impairs endoderm differentiation and causes apoptosis of epiblast cells. The endoderm defects have been attributed to the loss of HNF4, disabled-2, and GATA-4. However, the mechanisms underlying epiblast apoptosis are unclear. In this study we used mouse embryonic stem cell–derived embryoid bodies (EBs) as a model for peri-implantation development and found that ablation of GATA-6 causes massive apoptosis during EB differentiation. Endoderm grafting experiments and ectopic basement membrane (BM) assembly suggest that both BM and non-BM factors contribute to cell survival. Furthermore, the increased cell death in mutant EBs is accompanied by reduced expression of bone morphogenetic protein 2 (BMP-2). Chromatin immunoprecipitation reveals direct binding of GATA-6 to the Bmp2 promoter. Treatment of the mutant EBs with BMP-2 markedly suppresses apoptosis, whereas stable overexpression of the BMP antagonist noggin or a dominant-negative BMP receptor in normal EBs leads to increased apoptosis. Last, activation of SMAD1/5 by phosphorylation is significantly inhibited in the absence of GATA-6, and this is reversed by exogenous BMP-2. Treatment of normal EBs with SMAD phosphorylation inhibitor increases apoptosis. Collectively these results suggest that GATA-6 promotes cell survival by regulating endoderm expression of BMP-2 and BM during embryonic epithelial morphogenesis.

Adenosine diphosphate-ribosylation factors (Arfs) are a family of guanosine triphosphate-binding proteins involved in fundamental biological processes including secretion, endocytosis, phagocytosis, cytokinesis, cell adhesion and tumor cell invasion. We report here the molecular cloning, chromosome localization and expression analysis of porcine Arf1-6, of which Arf1-3 (Class I) have >93% similarity to each other and encode nearly similar proteins with 181 amino acids in length. Arf4 and Arf5 (Class II) are 78-81% homologous to Class I Arfs and both encode a protein of 180 amino acids, Arf6 (Class III) shows 64-68% homology to the other Arfs and encodes 175 amino acids. With radiation hybrid mapping, porcine Arf1-6 are assigned to chromosomes 14q21-q22, 12p14, 5p12-q11, 13q21.1, 18q24, 1q21-q27, respectively. Moreover, real-time quantitative RT-PCR assays show that porcine Arf1-6 are ubiquitous in all tissues examined, with the highest levels in the kidney and stomach and the lowest in muscle and the heart. This is the first report of molecular characterization of the Arf gene family in pigs.

Summary Nutrients are not only organic compounds fueling bioenergetics and biosynthesis, but also key chemical signals controlling growth and metabolism. Nutrients enormously impact the production of reactive oxygen species (ROS), which play essential roles in normal physiology and diseases. How nutrient signaling is integrated with redox regulation is an interesting, but not fully understood, question. Herein, we report that superoxide dismutase 1 (SOD1) is a conserved component of the mechanistic target of rapamycin complex 1 (mTORC1) nutrient signaling. mTORC1 regulates SOD1 activity through reversible phosphorylation at S39 in yeast and T40 in humans in response to nutrients, which moderates ROS level and prevents oxidative DNA damage. We further show that SOD1 activation enhances cancer cell survival and tumor formation in the ischemic tumor microenvironment and protects against the chemotherapeutic agent cisplatin. Collectively, these findings identify a conserved mechanism by which eukaryotes dynamically regulate redox homeostasis in response to changing nutrient conditions.

Cyclin-dependent kinase 5 (CDK5) is a serine/threonine kinase homologue attributed to the mitotic cyclin-dependent kinase family. Both the kinase activity and the biological effects of CDK5 in central nervous system are mainly dependent on association with its regulatory subunit 1 known as CDK5R1 (p35). In the present study, the full-length coding regions of CDK5 and CDK5R1 were cloned from pigs. Radiation hybrid mapping localized porcine CDK5 to chromosome 18q12-13, whereas CDK5R1 was electro-localized to chromosome 12q12. Real-time quantitative RT-PCR (qRT-PCR) showed that CDK5 mRNA is ubiquitously present in all porcine tissues examined, with relatively high levels in cerebral cortex, cerebellum, testicle and lung. We also examined the expression profile of porcine CDK5/CDK5R1 in various tissues at different developmental stages. The results indicated that CDK5 mRNA reaches the highest level in cerebral cortex at two months of age and in cerebellum and liver at 4 months of age, respectively, whereas the peak level of CDK5R1 was observed in both cerebral cortex and cerebellum at two months of age, indicating the pivotal role of CDK5/CDK5R1 during the development of porcine brain.

CREB3L4 (cAMP response element binding protein3 like 4) belongs to mammalian CREB/ATF subfamily transcription factors featuring a unique putative transmembrane domain lined to the bZIP region in their C-terminals. We report here the cloning of porcine CREB3L4 and its alternative splicing variant. The open reading frame of normal CREB3L4 covers 1,188 bp and encodes a 395-amino acid polypeptide, whereas its variant encodes a C-terminal truncated protein comprising only 271 amino acids. The genomic sequence of porcine CREB3L4 spans approximately 6 kb consisted of ten exons and nine introns, and maps closely to Jumping translocation breakpoint gene in a head-to-head manner on the q arm of Sus Scrofa chromosome 4. Tissue expression analysis by RT-PCR indicates that normal porcine CREB3L4 is ubiquitously expressed with relatively high abundance in stomach, liver, cerebellum, and is a predominant form in various tissues, whereas the splicing variant is expressed at extremely low level in all examined tissues. Our study will lay the groundwork for the further investigation on the physiological function of CREB3L4 in pig models.

Recent evidence suggests that apoptosis of endothelial cells contributes to lumen formation during angiogenesis, but the biological mechanism remains obscure. In this study, we investigated the effect of nerve growth factor (NGF), a member of the neurotrophin family and a potential angiogenic factor, on human umbilical vein endothelial cells (HUVEC) apoptosis and the formation of lumen-like structures (LLS) by cultured HUVEC on Matrigel. We demonstrate that NGF induces cell apoptosis. NGF treatment has no significant effect on the expression level of its two receptors, TrkA and p75NTR. Blockade of both TrkA and p75NTR, but not that of either receptor alone significantly decreases NGF-induced cell apoptosis. NGF significantly increases formation of LLS which consist substantially of apoptotic cells. Application of NGF-neutralizing antibody or simultaneous blockade of TrkA and p75NTR significantly blocks spontaneous and NGF-induced LLS formation. These data support a role for NGF-induced cell apoptosis in LLS formation in vitro.

Objectives: The goal of this study was to evaluate the efficacy and the mechanism of 17β-estradiol-eluting stents on inhibiting neointimal formation of abdominal aortas in rabbits fed with high fat diet. Background: Animal experiments have shown that local delivery of estrogen reduced neointimal formation after coronary angioplasty. Preliminary trial of estrogen-eluting stent implantation in patients with coronary disease also suggests a reduction in restenosis. Methods: We implanted 17β-estradiol-eluting stents, or control phosphorylcholine- coated stents, or bare metal stents of abdominal aortas in rabbits fed with high fat diet. Histology, immunohistochemistry, and Western blot analysis were used to assess the efficacy and mechanism of inhibiting neointimal formation of 17β-estradiol-eluting stents. Results: Western blot analysis revealed marked increase in ERK phosphorylation in 30 min after deployment of phosphorylcholine-coated or bare metal stents, indicating activation of MAP kinase pathway. Immunohistochemistry showed intense staining of phospho-ERK in the medial smooth muscle cells in stent-implanted region. Extensive neointimal hyperplasia developed 12 weeks after stenting. In contrast, we observed significant inhibition of ERK phosphorylation and neointimal thickening in estrogen-eluting stent-implanted animals. Immunohistochemistry of factor VIII-related antigen demonstrated an accelerated reendothelialization as compared with the bare metal stent or phosphorylcholine-coated stent-implanted controls. Conclusions: Current study suggests that estrogen-eluting stents reduce neointimal formation and hence prevent restenosis after angioplasty possibly by inhibiting ERK activation in smooth muscle cells and promoting reendothelialization. © 2007 Wiley-Liss, Inc.

Sensing force is crucial to maintain the viability of all living cells. Despite its fundamental importance, how force is sensed at the molecular level remains largely unknown. Here we show that stomatin-like protein-3 (STOML3) controls membrane mechanics by binding cholesterol and thus facilitates force transfer and tunes the sensitivity of mechano-gated channels, including Piezo channels. STOML3 is detected in cholesterol-rich lipid rafts. In mouse sensory neurons, depletion of cholesterol and deficiency of STOML3 similarly and interdependently attenuate mechanosensitivity while modulating membrane mechanics. In heterologous systems, intact STOML3 is required to maintain membrane mechanics to sensitize Piezo1 and Piezo2 channels. In C57BL/6N, but not STOML3−/− mice, tactile allodynia is attenuated by cholesterol depletion, suggesting that membrane stiffening by STOML3 is essential for mechanical sensitivity. Targeting the STOML3–cholesterol association might offer an alternative strategy for control of chronic pain., To maintain viability, cells must be able to sense and respond to mechanical stimuli. Here, Qi et al. show that the STOML3 protein acts in mechanosensation by binding cholesterol and regulating membrane stiffness which can in turn regulate ion flux through mechanosensitive channels.

Background Bristol stool form 1 and 2 is an important predictor of inadequate bowel preparation. Aim To evaluate the efficacy of supplemental preparation in bowel cleansing quality among patients with Bristol stool form 1 and 2, as well as the feasibility of tailored bowel preparation guided by Bristol stool form scale. Methods Patients with Bristol stool form 1 and 2 from 3 Chinese tertiary hospitals randomly received either 2 L PEG-ELP (group A) or 10 mg bisacodyl plus 2 L PEG-ELP (group B); patients with Bristol stool form 3 to 7 received 2 L PEG-ELP (group C) for bowel preparation. The primary endpoint is the rate of adequate bowel reparation for the whole colon. The adequate bowel preparation rate for separate colon segments, the polyp detection rate (PDR), tolerability, acceptability, sleeping quality and compliance were evaluated as secondary endpoints. Results 700 patients were randomized. In per-protocol analysis, patients in group B attained significantly higher successful preparation rate than group A (88.7% vs. 61.2%, p