Telomere length and micronuclei trajectories in APP/PS1 mouse model of Alzheimer's disease: Correlating with cognitive impairment and brain amyloidosis in a sexually dimorphic manner.

Although studies have demonstrated that genome instability is accumulated in patients with Alzheimer's disease (AD), the specific types of genome instability linked to AD pathogenesis remain poorly understood. Here, we report the first characterization of the age- and sex-related trajectories of telomere length (TL) and micronuclei in APP/PS1 mice model and wild-type (WT) controls (C57BL/6). TL was measured in brain (prefrontal cortex, cerebellum, pituitary gland, and hippocampus), colon and skin, and MN was measured in bone marrow in 6- to 14-month-old mice. Variation in TL was attributable to tissue type, age, genotype and, to a lesser extent, sex. Compared to WT, APP/PS1 had a significantly shorter baseline TL across all examined tissues. TL was inversely associated with age in both genotypes and TL shortening was accelerated in brain of APP/PS1. Age-related increase of micronuclei was observed in both genotypes but was accelerated in APP/PS1. We integrated TL and micronuclei data with data on cognition performance and brain amyloidosis. TL and micronuclei were linearly correlated with cognition performance or Aß40 and Aß42 levels in both genotypes but to a greater extent in APP/PS1. These associations in APP/PS1 mice were dominantly driven by females. Together, our findings provide foundational knowledge to infer the TL and micronuclei trajectories in APP/PS1 mice during disease progression, and strongly support that TL attrition and micronucleation are tightly associated with AD pathogenesis in a female-biased manner.

Activation of Wnt/ß-Catenin signaling in EpCAMhigh/CD44+ cells endow colorectal cancer with tumor proliferation and oxaliplatin chemoresistance.

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. 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/ß-catenin signaling pathway is activated in EpCAMhigh/CD44+ HCT116 cells. CONCLUSION: Activation of Wnt/ß-Catenin signaling in EpCAMhigh/CD44+ cells endow colorectal cancer with tumor proliferation and oxaliplatin chemoresistance.

Postsurgical Latent Pain Sensitization Is Driven by Descending Serotonergic Facilitation and Masked by µ-Opioid Receptor Constitutive Activity in the Rostral Ventromedial Medulla.

Following tissue injury, latent sensitization (LS) of nociceptive signaling can persist indefinitely, kept in remission by compensatory µ-opioid receptor constitutive activity (MORCA) 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-HT3R antagonist ondansetron; the 5-HT2AR antagonist MDL-11 939 had no effect. Second, to test for MORCA, we microinjected the MOR inverse agonist d-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) and/or neutral opioid receptor antagonist 6ß-naltrexol. Intra-RVM CTAP produced mechanical hypersensitivity at both hindpaws; 6ß-naltrexol had no effect by itself, but blocked CTAP-induced hypersensitivity. This indicates that MORCA, 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-HT3R signaling in the dorsal horn, and this LS is tonically opposed by MORCA in the RVM. The 5-HT3 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-HT3 receptor signaling at the spinal cord blocks behavioral signs of postsurgical latent sensitization. We conclude that MORCA in the RVM opposes descending serotonergic facilitation of LS and that the 5-HT3 receptor is a promising therapeutic target for the development of drugs to prevent the transition from acute to chronic postsurgical pain.

Genetic and functional evidence for gp130/IL6ST-induced transient receptor potential ankyrin 1 upregulation in uninjured but not injured neurons in a mouse model of neuropathic pain.

ABSTRACT: 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.

A comprehensive study of the genotoxic and anti-genotoxic effects of homocysteine in HUVECs and mouse bone marrow cells.

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.

CREG1 promotes lysosomal biogenesis and function.

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.

Short-term in vitro glutamine restriction differentially impacts the chromosomal stability of transformed and non-transformed cells.

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.

PTEN suppresses epithelial-mesenchymal transition and cancer stem cell activity by downregulating Abi1.

The epithelial-mesenchymal transition (EMT) is an embryonic program frequently reactivated during cancer progression and is implicated in cancer invasion and metastasis. Cancer cells can also acquire stem cell properties to self-renew and give rise to new tumors through the EMT. Inactivation of the tumor suppressor PTEN has been shown to induce the EMT, but the underlying molecular mechanisms are less understood. In this study, we reconstituted PTEN-deficient breast cancer cells with wild-type and mutant PTEN, demonstrating that restoration of PTEN expression converted cancer cells with mesenchymal traits to an epithelial phenotype and inhibited cancer stem cell (CSC) activity. The protein rather than the lipid phosphatase activity of PTEN accounts for the reversal of the EMT. PTEN dephosphorylates and downregulates Abi1 in breast cancer cells. Gain- and loss-of-function analysis indicates that upregulation of Abi1 mediates PTEN loss-induced EMT and CSC activity. These results suggest that PTEN may suppress breast cancer invasion and metastasis via dephosphorylating and downregulating Abi1.

PTEN dephosphorylates Abi1 to promote epithelial morphogenesis.

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.

Impaired mechanical, heat, and cold nociception in a murine model of genetic TACE/ADAM17 knockdown.

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 ß-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., Mitric, 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.

SOD1 Phosphorylation by mTORC1 Couples Nutrient Sensing and Redox Regulation.

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.

Identification of Chloride Channels CLCN3 and CLCN5 Mediating the Excitatory Cl- Currents Activated by Sphingosine-1-Phosphate in Sensory Neurons.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid involved in numerous physiological and pathophysiological processes. We have previously reported a S1P-induced nocifensive response in mice by excitation of sensory neurons via activation of an excitatory chloride current. The underlying molecular mechanism for the S1P-induced chloride conductance remains elusive. In the present study, we identified two CLCN voltage-gated chloride channels, CLCN3 and CLCN5, which mediated a S1P-induced excitatory Cl- current in sensory neurons by combining RNA-seq, adenovirus-based gene silencing and whole-cell electrophysiological voltage-clamp recordings. Downregulation of CLCN3 and CLCN5 channels by adenovirus-mediated delivery of shRNA dramatically reduced S1P-induced Cl- current and membrane depolarization in sensory neurons. The mechanism of S1P-induced activation of the chloride current involved Rho GTPase but not Rho-associated protein kinase. Although S1P-induced potentiation of TRPV1-mediated ionic currents also involved Rho-dependent process, the lack of correlation of the S1P-activated Cl- current and the potentiation of TRPV1 by S1P suggests that CLCN3 and CLCN5 are necessary components for S1P-induced excitatory Cl- currents but not for the amplification of TRPV1-mediated currents in sensory neurons. This study provides a novel mechanistic insight into the importance of bioactive sphingolipids in nociception.

Microgravity simulation activates Cdc42 via Rap1GDS1 to promote vascular branch morphogenesis during vasculogenesis.

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.

Randomized controlled trial: Standard versus supplemental bowel preparation in patients with Bristol stool form 1 and 2.

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<0.001) and similar with group C (88.7% vs. 85.0%, p = 0.316). The PDR in group B was significantly higher than group A (43.2% vs. 25.7%, p<0.001). Acceptability was much higher in group B and C. CONCLUSIONS: 10 mg bisacodyl plus 2 L PEG-ELP can significantly improve both bowel preparation quality and PDR in patients with Bristol stool form 1 and 2. Bristol stool form scale may be an easy and efficient guide for tailored bowel preparation before colonoscopy.

Integrin and dystroglycan compensate each other to mediate laminin-dependent basement membrane assembly and epiblast polarization.

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-ß1 null EBs with induction of polarization at reduced efficiency. ß-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 ß1-null EBs, reducing BM and ablating cell polarization. When Lm111 was incubated with integrin-ß1/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.

Construction of interference vector targeting Ep-CAM gene and its effects on colorectal cancer cell proliferation.

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<0.01). Western blot analysis results showed that Ep-CAM protein expression was significantly lower in interference group compared with that of the empty plasmid group and the control group (P<0.01). MTT assay results demonstrated that the proliferation ability of cells in the interference group was significantly inhibited compared with the two other groups (P<0.05). CONCLUSION: Silencing of Ep-CAM can significantly inhibit the proliferation of colorectal cancer cells.

Bnip3 and AIF cooperate to induce apoptosis and cavitation during epithelial morphogenesis.

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-1ß, the common ß 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.

Molecular cloning, chromosomal localization and expression pattern of porcine ADP-ribosylation factor(Arf) gene family.

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.

Rac1 is essential for basement membrane-dependent epiblast survival.

During murine peri-implantation development, the egg cylinder forms from a solid cell mass by the apoptotic removal of inner cells that do not contact the basement membrane (BM) and the selective survival of the epiblast epithelium, which does. The signaling pathways that mediate this fundamental biological process are largely unknown. Here we demonstrate that Rac1 ablation in embryonic stem cell-derived embryoid bodies (EBs) leads to massive apoptosis of epiblast cells in contact with the BM. Expression of wild-type Rac1 in the mutant EBs rescues the BM-contacting epiblast, while expression of a constitutively active Rac1 additionally blocks the apoptosis of inner cells and cavitation, indicating that the spatially regulated activation of Rac1 is required for epithelial cyst formation. We further show that Rac1 is activated through integrin-mediated recruitment of the Crk-DOCK180 complex and mediates BM-dependent epiblast survival through activating the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway. Our results reveal a signaling cascade triggered by cell-BM interactions essential for epithelial morphogenesis.

[Construction and assessment of short-hairpin RNA eukaryotic expression vector targeting TGF-beta1 labeled by GFP].

AIM: To construct short hairpin RNA (shRNA) eukaryotic expression vectors targeting TGF-beta1 for further research on the effects of TGF-beta1 on vasculogenesis and angiogenesis. METHODS: Three pairs of siRNA target sequences coding from the mRNA of TGF-beta1 gene were designed and three pairs of nucleotides were synthesized. After annealing, the double-strand DNA products were ligated into the pEN_mH1c entry vector, and in turn into the shRNA eukaryotic expression vector pDS_hpEy labled by GFP through the LR recombination reaction. After sequencing successfully, the three resulting TGF-beta1 shRNA expression vectors were transfected into the mouse fibroblast cell line (NIH/3T3), and then cell clones stably expressing TGF-beta1 shRNA were screened. Reverse Transcript-Polymerase Chain Reaction (RT-PCR) and Western blot were used to detect the mRNA and protein expression. RESULTS: RT-PCR and Western blot showed that one of the TGF-beta1 shRNA expression vectors pDS_Tc downregulated TGF-pl mRNA and protein expression markedly in NIH/3T3 cells. CONCLUSION: ShRNA eukaryotic expression vectors targeting TGF-beta1 are successfully constructed which can be used for further investigation on the mechanism through which TGF-beta1 regulates vasculogenesis and angiogenesis.