FE) and photo-bias stability of the double-channel device were improved by increasing the thickness of the front IZO film (intt) compared to the single-ZTO-channel device. A high-mobility (approximately 32.3 cm2/Vs) ZTO/IZO transistor with excellent photo-bias stability was obtained from Sn doping of the front IZO layer. First-principles calculations revealed an increase in the formation energy of O vacancy defects in the Sn-doped IZO layer compared to the IZO layer. This observation suggests that the superior photo-bias stability of the double-channel device is due to the effect of Sn doping during thermal annealing. However, these improvements were observed only when intt was less than the critical thickness. The rationale for this observation is also discussed based on the oxygen vacancy defect model.This study examined the performance and photo-bias stability of double-channel ZnSnO/InZnO (ZTO/IZO) thin-film transistors. The field-effect mobility (μ Metal oxide thin-film transistors (TFTs) have attracted considerable attention for applications in the active-matrix (AM) backplanes of liquid crystal displays (LCDs) and organic light-emitting diode (OLED) displays due to their high mobility, low production cost, and low-temperature processing capability12362 or Al2O3, which were deposited by plasma-enhanced chemical vapor deposition (PECVD) or atomic layer deposition, were reported to effectively suppress the NBIS instability of the resulting metal oxide TFTs. This suppression was attributed to the large hole injection barrier (>2.0 eV) and low interfacial trap density of these insulatorsOV) defects is related to the intrinsic nature of metal oxide semiconductor materials. The incorporation of carrier suppressors, such as Zr, Hf, and Ga, to the relevant metal oxide semiconductor can help reduce the NBIS instability because these suppressors can effectively reduce the concentration of OV defects in the resulting channel layer. However, the field-effect mobility of the resulting oxide TFTs was adversely affected by the addition of such suppressors11High-quality gate insulators, such as SiOOV defects under In-rich conditionsThe incorporation of In cations to multi-component ZnO-based channels is one of best ways to enhance the field-effect mobility of the resulting oxide TFTs2 gate insulator on the mobility and positive-bias stress (PBS) instability of the resulting oxide TFTs. The improvement of both the mobility and PBS stability of IGZO/GZO TFTs was attributed to the localization effect of the conducting channel path, where the inserted conducting GZO layer reduced the screening length of the channel layer under an on-state current condition while suppressing the trap-induced PBS instability at the channel bulk and back surface regionSeveral studies have assessed the benefits of a double-channel structure15161718FE) of 32.3 cm2/Vs, a low subthreshold gate swing (SS) of 0.12 V/decade, a reasonable threshold voltage (Vth) of 0.5 V, a high Ion/off ratio of > 108, and the best NBIS stability. A depth profile analysis of the channel stack revealed the inter-diffusion of Sn and In atoms, which occurs during thermal annealing at 500°C. Therefore, the front IZO layer was doped with Sn cations. The microscopic origin for such improvement was examined by first-principles calculations based on density functional theory. The formation energy of oxygen vacancies for the IZO front layer substantially increased (by approximately 0.3 eV) due to Sn doping, which is responsible for the improvement in the NBIS stability of the double-channel device.In this study, double-channel oxide TFTs consisting of a back-layer ZTO film and front-layer IZO film were fabricated. The thickness-dependent performance and stability of the front IZO layer were examined experimentally. The optimum device was achieved for the 5-nm-thick IZO device, which exhibited a high field-effect mobility (μintt = 5.0 nm), and ZTO/IZO (intt = 6.3 nm) devices. μFE was determined by the maximum trans-conductance at a drain voltage (VDS) of 0.1 V, and Vth was determined from the gate voltage (VGS) required to produce a drain current of L/W × 10 nA at VDS = 5.1 V. The subthreshold gate swing (SS = dVGS/dlogIDS) was extracted from the linear portion of a plot of the log IDS versus VGS. For the first-series devices, the front IZO layer was prepared at an oxygen ratio ([O2]/[Ar + O2]) of 0.3. The single ZTO control device exhibited a μFE, SS, Vth, and Ion/off ratio of 28.8 cm2/Vs, 0.20 V/decade, 1.4 V, and 1.5 × 108, respectively. The transporting properties of the double-channel devices were improved substantially by increasing the thickness of the front IZO layer, as summarized in FE value obtained from the ZTO/IZO (intt = 6.3 nm) devices was 33.0 cm2/Vs. The enhanced transporting properties of the double-channel device were reflected in the superior output characteristics, as shown in th of the double-channel devices were displaced to the negative direction with increasing intt. This result indicates that the number of free electron carriers (Nd) in the channel layer is proportional to the thickness of the front IZO layer. The high mobility of the double-channel devices was attributed to the increasing In content caused by the intercalation of In atoms with a larger radius1th, the ZTO/IZO (intt = 5.0 nm) device was found to be optimal: a low voltage of 0.41 V is desirable for low-power-consumption AM panels. A similar behavior was observed at a higher oxygen ratio ([O2]/[Ar + O2]) of 0.4, as listed in it,max) according to the following equationB is Boltzmann's constant, T is the absolute temperature, q is the electron charge, and Ci is the gate capacitance per unit area. The Dit,max of the resulting devices decreased as intt was increased for an oxygen ratio of 0.3 and 0.4, respectively. This increase strongly suggests that the interfacial properties between the SiO2 gate insulator and oxide semiconductor channel layer were improved by inserting a front IZO layer. The physical implication will be discussed later in conjunction with the intt-dependent bias and photo-bias stability.The SS value of a given TFT device is related to the interfacial trap density (Dintt = 5.0 nm) devices. The devices were stressed under the following conditions: VGS and VDS were set to 20 V and 5.1 V, respectively, at room temperature, and the stress duration was 7,200 s. For the control device, a parallel Vth shift of 2.2 V to a higher voltage with increasing PBS time was observed without an accompanying change in μFE, SS, and the Ion/off ratio. The Vth stability of the IGZO TFTs under PBS was clearly improved by adopting a double-channel structure (see th shift was further suppressed to ~0.2 V for ZTO/IZO (intt = 6.3 and 8.3 nm) devices.The effects of the PBS and negative gate bias stress (NBS) on the transfer characteristics of both devices were investigated. ture see . It is nGS of −20 V and a stress duration of 7,200 s. Again, the NBS-induced variation was reduced significantly from −0.7 V (control device) to −0.3 V for the double-channel device, as shown in th variations of the ZTO/IZO devices prepared at an oxygen ratio of 0.3. The PBS-induced Vth displacement decreased significantly with increasing intt suggesting that the front IZO film provides a rapid carrier path and lower effective trap density. The Vth instability by the application of NBS was also suppressed by increasing intt, except for intt ≥ 6.3 nm.The effect of the channel structure on the NBS instability of the resulting TFTs was also investigated. The devices were stressed under a V2 was used under identical NBS conditions. Although the NBIS instability for each device was worse than the dark NBS instability, the changes in Vth by the application of NBIS were similar to those by NBS. When intt was less than the critical thickness (critt), the NBIS stability of the double-channel device improved with increasing intt. Here, critt is defined as the maximum thickness that results in higher mobility and better photo-bias stability compared to the single-channel device. For example, the negative Vth shift of the double-channel device (intt = 5 nm) was reduced from 12.5 V (single ZTO device) to 4.1 V (see intt ≤ critt) exhibited superior photo-bias stability, and the resulting oxide TFTs exhibited improved transporting properties compared to the control device. This result is difficult to understand because higher-In-containing oxide TFTs suffer from severe NBIS degradation due to the easy formation of oxygen vacancy defect centers. However, when intt > critt, the NBIS stability as well as NBS stability of the resulting double-channel device deteriorated abruptly. Therefore, the 6.3-nm-thick device fabricated at an oxygen ratio of 0.3 suffered from the most severe degradation (ΔVth = −15.2 V), which is larger than that of the ZTO control device, as shown in The photo-bias instability of the ZTO and ZTO/IZO devices was compared, as shown in .1 V see . The douintt = 5 nm) was much lower than that of the single-channel device. Here, the extracted DOS distributions are the summation of the bulk trap states (NSS) in the channel region and Nit at the channel/gate insulator interface. Because the thickness of the back ZTO channel was fixed to 35 nm for all devices, the NSS values for both devices would be comparable. Therefore, the In-rich composition can effectively eliminate the Nit at the IZO/gate insulator interface compared to those at the ZTO/gate insulator interface, which is consistent with the Nit,max variations extracted from the SS values.To obtain more insight into these anomalous behaviors, the DOS distributions of the devices in this study were calculated using the Meyer-Neldel ruleintt values. The MNR-derived DOS energy range was limited from the conduction band edge (EC) to (EC −0.4 eV), which corresponds to the tailing state of acceptor-like traps. Because the quasi-Fermi energy (EF) at the channel interface is positioned near the EC edge under the PBS condition, this DOS distribution directly affects the PBS instability, as discussed earlier. In contrast, NBS and NBIS instability mainly results from the VO deep state. Therefore, a complete understanding of photo-bias instability requires the comparative characterization of the VO density by X-ray photoelectron spectroscopy (XPS) and the theoretical electron calculation of VO defects.The PBS instability can be understood based on the carrier trapping mechanism. The amount of charge trapped into the channel bulk and at the channel/insulator interface will be proportional to the number of trap states regardless of the charge polarity. Therefore, the improvement in the PBS stability of the double structure devices can be attributed to the effective decrease in the interfacial trap density compared to that of the single-channel device. This inverse proportionality between the gate bias instability and DOS distribution was observed for all 2 stack. The Sn and In atoms in the channel stack region inter-diffused during thermal annealing for 1 h at 500°C, suggesting that the front IZO layer was doped with Sn cations. Pre-existing OV defects in the channel layer have a deleterious effect on the NBIS-induced instabilityOV) to the double-charged oxygen vacancy defects (OV+2), which results in the delocalized electrons in the conduction band. The influence of Sn on the oV formation energy was examined by first-principles calculations using the VIENNA ab initio simulation package (VASP)k-point mesh were used. For the exchange-correlation energy of electrons, the generalized-gradient approximation (GGA) + U calculation was used because it partially corrects the band-gap problem in GGA by decreasing the repulsion between oxygen p and metal d statesU parameters were set to 7.0, 3.5, and 8.0 eV for the In 4 d, Sn 4 d, and Zn 3 d states, respectively.Thus, the atomic distribution and chemical state of a ZTO/IZO film were analyzed by XPS. a-ITZO) compound with a stoichiometry of nIn:nSn:nZn = 1:1:1 was modeled by a first-principles molecular dynamics (MD) simulation. The eight formula units of In2Sn2Zn2O9 were distributed randomly into a cubic supercell. The structure was heated to 2,000 K for 5 ps, and then, the temperature was reduced rapidly to 300 K at a −300 K/ps quenching speed. The lattice parameters and atomic positions were fully relaxed, resulting in the final amorphous structure shown in The amorphous structure of the InSnZnO (f) for OV defects in the neutral state was calculated using the following formula: totE(OV) and OV, respectively, and μO is the chemical potential of oxygen under the given growth condition. In the present study, The formation energy (EfE(OV) depends strongly on the local environment of oxygenOV configurations exist in the amorphous structure. Therefore, 33 different OV configurations were calculated by removing an oxygen atom in the modeled a-ITZO and examining how the neighboring cation species affect the formation energy of the OV. In Sn). The formation energies increase substantially with increasing NSn, indicating that OV becomes difficult to form as more Sn atoms are introduced to the material. This dependence is consistent with the previous first-principles study that demonstrated the higher formation energy of OV in crystalline SnO2 compared to those of In2O3 and ZnOIn an amorphous multicomponent oxide, intt < critt) can be attributed to the suppression of OV defects in the front IZO layer by Sn doping and the reduced Nit density at the front layer/gate insulator interface.In addition, a recently proposed model on the peroxide formation through the hole creation inside the valence band may be valid in the present case and may also contribute to the observed photo instabilityintt > critt) suffers from inferior photo-bias stability compared to that of the single-channel device. OV is reduced due to the weak bonding of In and oxygen.The remaining question is why the thick front channel device  exhibited poorer NBIS stability due to the large OV concentration of the Sn-free front IZO layer. The extreme case corresponds to the single-IZO-channel device. The oxide TFTs with a 35-nm-thick single IZO channel did not exhibit any Ion/off modulation but instead exhibited simple metallic behavior. The Nd value of the 35-nm-thick IZO film was determined to be > 1020 cm−3 from the Hall effect measurement, which is consistent with the thick IZO film (intt > critt) with a high OV concentration.intt ≤ critt) and thick (intt > critt) IZO devices. There are two competing factors governing the PBS, NBS, and NBIS instability of the double-channel devices. One factor is the lower Dit,max between the Sn-doped IZO (IZTO) and SiO2 insulator compared to that between the ZTO and SiO2 insulator. The other factor is the creation of OV defects. For thin (intt ≤ critt) devices, the suppressed OV defect and lower Dit,max values are responsible for the improvement in the gate bias and photo-bias stability. However, thick (intt > critt) devices suffer from the abrupt creation of OV in the interfacial In-rich region due to the limited Sn diffusion to the IZO front layer, which leads to the deterioration of the NBS and NBIS stability.int dependency, the relative band structure for the ZTO/IZO/SiO2 stack was further analyzed by XP spectra using a monochromatic Al-Kα source. The XP valence band (VB) profiles near the bandgap for the ZTO, 5.0-nm-thick IZO, 6.3-nm-thick IZO, and SiO2 are depicted in 2, respectively. The band diagrams obtained from the VB spectra and optical bandgaps of the IZO and ZTO films are depicted in To confirm the proposed origin of the improved mobility and photo-bias stability with the strong tintt > critt) indeed has a high Nd due to the high OV concentration. The critt value of the double-channel TFTs clearly depends on the content of OV defect in a front layer. Indeed, the critt value for the double-channel TFTs increased from 5.0 to 6.5 nm when the oxygen ratio of O2/[O2 ± Ar] during the sputtering of the IZO layer increased from 0.3 to 0.4, as shown in O formation in the In-rich interfacial region due to the limited diffusion length of the Sn cation. In previous studies, the thickness of the front layer for the optimized double-channel device was in range of 3.5–5.0 nm18This remarkable difference suggests that the thicker IZO film  suffered from inferior NBS/NBIS stability and a large negative Vth displacement compared to those of the ZTO control device. This poor performance is attributed to the limited diffusion length of the Sn atoms compared to the thickness of the thick front IZO layer during thermal annealing. Therefore, careful control of the intt for the interfacial layer is essential for designing a double-channel device that exhibits improved performance and bias stability compared to the single-channel device. Overall, double-channel ZTO/IZO TFTs can be used as the backplane electronics for high-resolution and large-sized AMOLED and TFT-LCD displays.Double-channel ZTO/IZO TFTs exhibited improved mobility and photo-bias stability compared to single-channel ZTO TFTs when the front IZO thickness was less than the critical thickness. These intriguing properties were partially attributed to the reduced D2 thin film as a gate insulator was deposited onto a patterned Mo (200-nm-thick)/glass substrate by PECVD at 380°C. Front IZO thin films with intt values of 2.5–8.3 nm were deposited onto SiO2/Mo/glass substrates by DC sputtering. The working pressure was 0.26 Pa, and the relative oxygen ratio ([O2]/[Ar + O2]) was varied from 0.30 to 0.40. The DC power density of the InZnO (IZO) target (50 wt% In) was fixed to 2.2 W/cm2, whereas the deposition time was varied from 30 to 240 s. A 35-nm-thick ZTO film (back layer) was deposited onto an IZO/Mo/glass substrate. The oxygen flow rate of [O2]/[Ar + O2] was fixed to 0.10 for all devices. The Zn:Sn composition was approximately 50:50. The active channel was defined using a shadow mask during ZTO/IZO channel formation, and the ITO source/drain (S/D) electrode was deposited using the same sputtering system. The fabricated TFTs had a bottom gate structure with a channel width (W) and length (L) of 1,000 μm and 150 μm, respectively. The devices were annealed in air for 1 h at 500°C. The transfer characteristics of the oxide TFTs were measured using a Keithley 2636 Source Meter at room temperature. The film thickness was determined by ellipsometry and confirmed by X-ray reflectivity measurements. The cation compositions and chemical states of the ZTO and IZO films were examined by X-ray fluorescence spectroscopy  and XPS, respectively.A 120-nm-thick SiOH.Y.J., J.-U.B. and W.-S.S. designed this work. J.K.J. and S.H. wrote the manuscript. H.Y.J., A.Y.H. and D.-H.K. performed the experiments and electrical measurements. C.K.L. contributed to the XPS analysis. Y.K. and S.H. performed the theoretical calculations. All authors discussed the results and commented on the manuscript. The project was supervised by J.K.J.SI


Diet-induced obesity (DIO) is a significant health concern which has been linked to structural and functional changes in the gut microbiota. Exercise (Ex) is effective in preventing obesity, but whether Ex alters the gut microbiota during development with high fat (HF) feeding is unknown.Determine the effects of voluntary Ex on the gastrointestinal microbiota in LF-fed mice and in HF-DIO.Male C57BL/6 littermates (5 weeks) were distributed equally into 4 groups: low fat (LF) sedentary (Sed) LF/Sed, LF/Ex, HF/Sed and HF/Ex. Mice were individually housed and LF/Ex and HF/Ex cages were equipped with a wheel and odometer to record Ex. Fecal samples were collected at baseline, 6 weeks and 12 weeks and used for bacterial DNA isolation. DNA was subjected both to quantitative PCR using primers specific to the 16S rRNA encoding genes for Bacteroidetes and Firmicutes and to sequencing for lower taxonomic identification using the Illumina MiSeq platform. Data were analyzed using a one or two-way ANOVA or Pearson correlation.2 = 0.35, p = 0.043).HF diet resulted in significantly greater body weight and adiposity as well as decreased glucose tolerance that were prevented by voluntary Ex (p<0.05). Visualization of Unifrac distance data with principal coordinates analysis indicated clustering by both diet and Ex at week 12. Sequencing demonstrated Ex-induced changes in the percentage of major bacterial phyla at 12 weeks. A correlation between total Ex distance and the ΔCt Bacteroidetes: ΔCt Firmicutes ratio from qPCR demonstrated a significant inverse correlation (rEx induces a unique shift in the gut microbiota that is different from dietary effects. Microbiota changes may play a role in Ex prevention of HF-DIO. Approximately 35% of adults Recent research indicates that the gut microbiota induces the development of obesity in both humans While the relationship between diet-induced changes in gut microbiota may be model dependent, “microbiota transplant” models using germ-free mice demonstrated that gut microbes from obese mice have a direct effect in increasing fat mass and body weight compared to introduction of microbes from lean mice Exercise (Ex) is known to confer numerous health-related benefits. Recent studies indicate that Ex can normalize body weight, body fat, hyperglycemia and markers of inflammation in mouse models of diet-induced obesity (DIO) n-butyrate concentration as a consequence of a corresponding change in the n-butyrate producing bacteria compared to sedentary rats. More recently, Choi et al. examined the effect of Ex on polychlorinated biphenyl metabolism and the gut microbiota. They found that Ex altered the gut microbiota in normal chow-fed mice, predominantly affecting the families Erysipelotrichaceae and EnterococcaceaeFew studies have specifically examined the effect of Ex on the gut microbiota. An early study by Matsumoto et al. examined the effect of chronic, load-bearing Ex on cecal microbiota in a non-obese rat model In this study, we tested the hypothesis that voluntary Ex would reshape the gut microbiota in both LF and HF-fed mice and that Ex would normalize the gut microbiota in HF mice at the phylum level, in addition to mitigating the development of HF-DIO. Our results indicate that HF diets do indeed promote increased fatness, hyperglycemia and an altered gut microbiota profile compared to LF fed mice. Voluntary Ex prevented these changes in HF mice and resulted in restoration of the phylotypic profile observed with LF feeding. There was a significant correlation between the distance run and alterations in the major gut bacterial phyla for the Ex mice. In addition, differences were also observed at the sub-phylum level between the LF and HF groups with Ex. These findings may offer new insights into the beneficial effect of regular Ex in preventing DIO by modifying the bacterial balance of the gut microbiota.ad libitum.All experimental protocols were approved by the Midwestern University Institutional Animal Care and Use Committee (IACUC) and all procedures were performed in accordance with Midwestern University's guidelines for the care and use of laboratory animals. Five week old, male C57BL/6 mice (n = 48)  were received as littermates (15–19 g) and allowed to acclimate in Midwestern University's animal facility for 1 week prior to their random assignment to one of 4 diet and activity treatment groups. During the acclimation period, mice were kept in their respective litters and provided standard chow  and reverse osmosis water Following the acclimation phase, mice were randomly assigned to diet and activity groups based on equal distribution of littermates . Dietary intake included either high fat diet  or low fat diet . Mice were individually housed in rat cages in the absence  or presence  of a non-load bearing 14.6 cm hamster wheel  suspended from a metal rod based on a design reported by Konhilas et al. Littermates were separated into groups as follows: LF/Sed, n = 12, LF/Ex, n = 10, HF/Sed, n = 14, and HF/Ex, n = 12. Two separate batches of mice were used: an initial batch of mice were studied for preliminary indications of a global microbiota shift ) and measurement of gut transit  and a second batch for quantitative PCR (qPCR) and sequencing studies . All mice were followed on the protocol for 12 weeks for collection of fecal samples and then an additional two weeks for collection of blood samples and harvesting of tissues.Initial, weekly and final body weights were determined for all mice. Twenty-four hour food intake was measured at week 12 of the experimental period. For these measurements, mice were placed in cages without bedding and each mouse was given one pre-weighed pellet of their respective diet, either LF or HF food, in a ceramic dish designed to minimize food loss or contamination by feces. After 24 hours, the pellet and its remnants were weighed to determine food consumption. Food intake was expressed as kcal/24 hours.Fasting blood glucose (FBG) and oral glucose tolerance (OGT) were measured at 13 weeks on the diet and activity program. Mice were fasted overnight in cages without bedding prior to measuring tail vein blood glucose levels using a OneTouch UltraMini® monitor . Oral glucose tolerance was measured immediately following the FBG test. Mice were gavaged with 25% glucose in saline (1 g/kg body weight) followed by blood glucose measurements at 15, 30, 60 and 120 minutes post glucose challenge 2 and then subjected to pneumothorax. Heart and soleus muscles were removed, rinsed several times, and blotted dry to determine their respective wet weights. Epididymal fat pads were excised, rinsed, blotted dry, weighed, and used as indicators of adipose tissue accumulation.At the end of week 13 of the protocol, mice were anesthetized with COad libitum. At 30 minutes prior to euthanasia, 0.1 ml of 0.5 mmol/L 70 kDa fluorescein isothiocyanate (FITC)–dextran  was administered by oral gavage Measurement of intestinal transit was performed as a terminal experiment. Mice were fasted for 16 h with water available Extraction of DNA from fecal bacteria followed the protocol of Wang et al. 5′-AGAGTT TGATCCTGGCTCAG-3′) with a 6′ carboxyfluorescein (6-FAM) label and 1492R (5′-GGTTACCTTGTTACGACTT-3′). Each 50 μl PCR reaction contained 50 ng of DNA, 5.0 μl 10× Taq Buffer, 4.0 μl dNTP mixture (2.5 mM each), 0.25 μl TaKaRa Ex Taq , 1 μl forward primer and 1 μl reverse primer (10 pmol/μl each). PCR was run for 30 cycles using the thermal profiles of: 1 min at 94°C; 30 cycles of ; and a final extension period of 6 min at 72°C. PCR products were verified by electrophoresis and visualization of a band at ∼1400 bp. Upon confirmation, the remaining PCR products were purified and stored at −80°C.The T-RFLP analysis was performed as previously described MspI enzyme (New England Biolabs Inc.) in a total volume of 15 μl  at 37°C for 3 hours. To enhance the fluorescent signal, each digested T-RFLP sample was desalted by single drop analysis. Digested samples were desalted, mixed with deionized formamide and 0.5 μl GeneScan-500 size standard (Applied Biosystems) followed by capillary electrophoresis . Fragment length and abundance were determined using GeneMapper software . Raw electropherograms were collected and analyzed for artifacts. To visualize relationships between the samples, sample distance values were analyzed by T-RFLP Analysis EXpedited (T-REX) and plotted using principal coordinates analysis (PCA) Purified 16S rDNA was digested using 20 U The two gut bacterial phyla of interest, Bacteroidetes and Firmicutes, were targeted by modifying a quantitative PCR assay developed by Armougom et al. using primer and probe sets that recognized a conserved region of the 16S rRNA encoding gene unique to these taxonomic groups The assay was performed on the ABI-PRISM 7700 Sequence Detection System . The methods on which this assay was based have been published previously Microbial sequencing was performed on the MiSeq Illumina platform  as previously described by Caporaso et al. Bioinformatic analysis of sequencing data was conducted using the QIIME 1.5.0 software suite http://metagenomics.anl.gov/linkin.cgi?project=7038) based at Argonne National Laboratory, including instant availability of the sequence data, bioinformatic analyses and tools.The data are publicly available via NCBI GenBank , a Pearson's r for correlation, a one-way ANOVA for qPCR data  and a two-way ANOVA for all other data comparisons on Prism 5  and SPSS 18  software. Two-way ANOVA results were reported as the F-statistics and p values and a Sidak post-hoc test was used for within group comparisons. To determine the significance of microbial community partitioning due to experimental treatments, permutational multivariate analysis of variance (PERMANOVA) was employed to generate p-values based on the observed Unifrac distance matrix relative to 10,000 randomly rearranged distance matrices. For display purposes only, the effect of exercise on the qPCR fold change in Bacteroidetes and Firmicutes in LF/Ex and HF/Ex mice is shown by the 2Mice in the Ex groups (LF/Ex and HF/Ex) spontaneously ran on the cage-mounted wheel and averaged 82±3.9 km/week and 89±7.9 km/week for the LF/Ex and HF/Ex groups, respectively p>0.05; . By the Metabolically, the HF/Sed mice demonstrated elevated body fat and altered glucose metabolism. Body fat, as measured by epididymal fat pad weight , was eleTwenty-four hour food intake, measured during week 13 of the protocol, was elevated by Ex  = 5.31, p = 0.032), but not diet  = 0.008, p = 0.93), with Ex influencing food intake of LF and HF mice similarly  = 2.17, p = 0.15; Diet and Ex did not significantly influence gastrointestinal transit under fasting conditions. Based on the geometric center measurements reported in Characterization of changes in the gut bacterial community as a consequence of diet and Ex was examined in three ways using bacterial 16S rRNA encoding genes present in the fecal pellets: terminal restriction fragment length polymorphism (T-RFLP), sequencing, and quantitative PCR (qPCR).T-RFLP was used to determine whether there was a global shift in the gut microbial community after the 12 week diet and activity protocol using the 16S rRNA encoding genes present in the fecal pellets. Subjective analysis of raw electropherograms from T-RFLP analysis demonstrated visible changes in the abundance and distribution of community groups as a result of diet and activity . PrincipCloser analysis of bacterial differences induced by diet and Ex were determined by sequencing the 16S rRNA encoding genes present in the fecal pellets. Principal coordinates analysis of Illumina MiSeq amplicon data demonstrated significant clustering at week 0 based on litter , which wSix different bacterial phyla were identified in the MiSeq 16S rRNA based amplicon libraries at the start and completion of the diet and Ex protocol . While there were no differences at baseline (data not shown), there were measureable differences in the percentage of these bacterial phyla among the 4 diet and activity groups by week 12 . For the−ΔΔCt values . As expected, there was no relationship between the data points determined for week 0 .As a test of the criterion validity of qPCR, a comparison was made between the qPCR and sequencing data. The ΔCt Bacteroidetes:ΔCt Firmicutes ratio determined by q-PCR and %Bacteroidetes:%Firmicutes ratio from sequencing was compared using a Pearson test on all data points from weeks 6 and 12. As shown in 2 = 0.35) based on a Pearson test .To validate the activity effect on bacterial balance, total Ex distance was correlated to the resultant ΔCt ratio of Bacteroidetes:Firmicutes determined using qPCR . The ratFamily Level Analysis. Family-level changes within the major phyla induced by the diet and Ex protocol were revealed by the sequencing data. Within the Firmicutes phylum, there were significant changes in the classes Clostridia, Bacilli, and Erysipelotrichi  = 13.76, p = 0.001), Lachnospiraceae  = 56.7, p<0.001) and Ruminococcaceae  = 37.38, p<0.001) in Sed and Ex mice. Ex significantly elevated the content of Lachnospiraceae  = 7.91, p = 0.011) and Ruminococcaceae  = 14.91, p = 0.001), with a tendency for an effect on Clostridiacea  = 3.17, p = 0.090). An interaction between diet and Ex was determined for Clostridiaceae  = 6.54, p = 0.019; LF/Ex compared to HF/Ex, p = 0.0015) and Ruminococcaceae  = 5.05, p = 0.036; activity had a greater effect in the HF fed mice compared to the LF mice). Lachnospiraceae had no interaction effect  = 1.72, p = 0.20).lotrichi . Three foccaceae . High faLactobacillaceae and Turicibacteraceae  = 11.27, p = 0.003), but there was no effect of diet  = 0.16, p = 0.69), although a trend for an interaction was detected  = 4.14, p = 0.055). HF/Sed mice demonstrated a bloom in Lactobacillaceae relative to the other treatment groups. Turicibacteraceae were significantly reduced by Ex in both LF and HF mice. High fat consumption decreased the percentage of Turicibacteraceae in the Sed and Ex groups  = 17.07, p<0.001; activity F = 5.20, p = 0.034), with a trend for an interaction effect  = 3.23, p = 0.087).The percentages of two different families within the class Bacilli were altered by the diet and activity protocol: teraceae . Ex decrErysipelotrichaceae was reduced by both HF diet and Ex  = 17.15, p<0.001; activity F = 6.17, p = 0.022), but Ex only exerted an effect in the LF fed mice  = 5.64, p = 0.03; p = 0.016 LF/Sed greater than LF/Ex).A third class of Firmicutes, Erysipelotrichi, was also altered by diet and activity . Within S24-7, S24-7  = 5.89, p<0.025), but Ex increased it  = 5.63, p = 0.028) equally in LF and HF mice  = 0.204, p = 0.66). Differences in the percentage of the phylum Actinobacteria based on the diet and activity protocol could be explained by differences in only one class (Actinobacteria), one order  and one family  = 6.45, p = 0.02; activity F = 4.86, p = 0.039), with Ex affecting only the LF condition  = 4.86, p = 0.039). Within the phylum Proteobacteria, a family in the Deltaproteobacteria class, Desulfovibrionaceae, demonstrated a trend toward a diet effect (Differences in the percentage of the phylum Bacteroidetes based on the diet and activity protocol could be explained primarily by differences in one class (Bacteroidia) and one family S24-7, . High fat effect   elevated levels of Firmicutes, Tenericutes, Proteobacteria, and Actinobacteria relative to the LF controls S24-7. High-fat sedentary mice had the lowest level of S24-7 family compared to the other treatment groups and the level was elevated by Ex. Exercise also led to an increase in butyrate-producing Firmicutes in fecal samples: Clostridiaceae, Lachnospiraceae and Ruminococcaceae. These bacteria are all members of Clostridiales order and are also linked to butyrate production n-butyrate in non-obese rats In the current study, Ex significantly increased the relative proportion of butyrate-producing bacteria such as Bacteroidales Lactobacillaceae in the HF mice, dropping it from greater than 20% in the HF/Sed mice to 0.35% in the HF/Ex mice. This dramatic reduction with Ex suggests an important role for Lactobacillaceae in sedentary lifestyle-associated weight gain. Lactobacilli are often used as probiotic agents. Depending on the Lactobacillus species and experimental model, the reported relationship between detectable Lactobacillus spp and the effects of Lactobacillus on weight gain and obesity can be quite different. Lactobacillus spp are reported to be elevated in obese children Lactobacillus reuteri is elevated, but L. paracasei and L. plantarum are reported to be reduced Lactobacillus spp given as probiotic therapy have dose-dependent effects with regards to weight changes L. plantarum has been reported to improve glucose sensitivity in response to a HF diet Exercise also reduced the percentage of the family The central questions raised by this study are, how does Ex alter the gut microbiota and does this contribute to the prevention of HF-DIO? Whether Ex operates to prevent HF-DIO through direct changes in the gut microbiota is an intriguing question because the answer could lead to new probiotics or prebiotics that provide for some of the benefits of Ex. The presence of a gut microbiota is known to help break down ingested food and likely allows for additional nutrient extraction not available to a germ free host Three factors determine the bacterial production of SCFA in the colon: the gut microbiota, availability of substrates, and intestinal transit time The amount of nutrients ingested could also impact bacterial content. In this study, Ex increased 24-hour caloric intake compared to Sed mice. Thus, microbial changes detected in the HF/Ex group beyond that of the HF/Sed could be related to even greater fat intake compared to HF/Sed or to an independent effect of Ex. A recent study by Simoes et al. demonstrated a change in the proportion of Bacteroidetes in monozygotic twins who were discordant with respect to diet An Ex-induced gut microbiota may increase fatty acid oxidation and utilization or decrease fat storage in the intestine. Exercise up-regulates AMP-activated protein kinase (AMPK) Another way in which Ex could exert a potentially beneficial effect through the gut microbiota is by suppressing inflammation. Exercise might prevent HF-DIO by blocking HF feeding-associated inflammation. Obesity and HF diets are linked to increased markers of endotoxemia and inflammation 2 = 0.33). This may be due to differences in the two techniques, including the methods of quantification (cycle number versus database-matching), universal primer sets used, and the specificity and inclusiveness of sequencing compared to qPCR. However, it can be seen in the One of the limitations of this study is that bacterial changes were only measured in fecal samples. Differences in the composition of the gut microbiota have been shown as one moves from the epithelial surface out into the lumen of the intestine Lactobacillaceae, Lachnospiraceae, and Ruminococcaceae and a decrease in the major constituent group of Bacteroidetes, S24-7. Exercise in the HF condition prevented some of the dietary changes, such as with Lactobacillaceae and S24-7, but intensified the blooms seen with HF feeding in others such as with Lachnospiraceae, and Ruminococcaceae. Further exploration of the gut microbiota changes induced by Ex may allow for exploitation of this effect and the development of treatments for obesity and dysbiosis associated with high fat intake.Exercise alters the gut microbiota in mice on both a LF and HF diet and normalizes major phylum-level changes for mice on the HF diet; furthermore, the volume of exercise  inversely correlates with the Bacteroidetes:Firmicutes ratio. The Ex-induced gut microbiota in both LF and HF conditions are different than their sedentary counterparts and yet, also different from each other. At the taxonomic level of bacterial family, HF diet induced blooms in some of the major constituent groups within Firmicutes such as Figure S1Identification of Amplicon Size for Bacteroidetes, Firmicutes and the Universal Primer Sets.(PDF)Click here for additional data file.Figure S2Diet and Activity Changed Bacterial Profiles Determined by T-RFLP and PCA.(PDF)Click here for additional data file.Figure S3Dendrogram Analysis of Diet and Activity Effects.(PDF)Click here for additional data file.Figure S4Diet and Activity Altered Major and Minor Bacterial Phyla.(PDF)Click here for additional data file.Figure S5Changes in the Bacteroidetes:Firmicutes Ratio Determined by qPCR and Sequencing.(PDF)Click here for additional data file.Table S1Primer and probe sets for Bacteroidetes and Firmicutes qPCR.(PDF)Click here for additional data file.Table S2Effect of diet and exercise on bacterial families with total contributions between 0% and 1.25%.(PDF)Click here for additional data file.File S1Commands used in the analysis of the sequencing dataset.(PDF)Click here for additional data file.


The Hepatitis C virus (HCV) core protein has been implicated as a potential oncogene or a cofactor in HCV-related hepatocellular carcinoma (HCC), but the underlying mechanisms are unknown. Overactivation of the Wnt/β-catenin signaling is a major factor in oncogenesis of HCC. However, the pathogenesis of HCV core-associated Wnt/β-catenin activation remains to be further characterized. Therefore, we attempted to determine whether HCV core protein plays an important role in regulating Wnt/β-catenin signaling in HCC cells.Wnt/β-catenin signaling activity was investigated in core-expressing hepatoma cells. Protein and gene expression were examined by Western blot, immunofluorescence staining, RT-qPCR, and reporter assay.c-Myc, cyclin D1, WISP2 and CTGF. Also, core protein increases cell proliferation rate and promotes Wnt3A-induced tumor growth in the xenograft tumor model of human HCC.HCV core protein significantly enhances Tcf-dependent transcriptional activity induced by Wnt3A in HCC cell lines. Additionally, core protein increases and stabilizes β-catenin levels in hepatoma cell line Huh7 through inactivation of GSK-3β, which contributes to the up-regulation of downstream target genes, such as HCV core protein enhances Wnt/β-catenin signaling activity, hence playing an important role in HCV-associated carcinogenesis. Hepatocellular carcinoma (HCC) ranks among the most common and deadly cancers worldwide HCV core is a 191 amino acid protein with RNA binding activity and may transactivate some transcription factors, such as NF-κB, AP-1 and SREBP APC, oncogenic mutations of β-catenin, upregulation of Frizzled type receptors (Fzds), and/or other Wnt signaling pathway alterations, play important roles in more than 33–67% of HCCs Aberrant activation of Wnt/β-catenin signaling results in enhanced cell growth and malignant cellular transformation. Inactivating mutations of In this study, we sought to investigate whether HCV core protein exerts any effect on Wnt/β-catenin signaling pathway and hence is involved in HCV-induced liver pathogenesis. We found that HCV core protein acts synergistically with Wnt3A on β-catenin-dependent transcriptional activity in HCC cell lines, and co-expression of core and Wnt3A induces stabilization and nuclear translocation of β-catenin, contributing to up-regulation of Wnt target gene expression. Cells transduced with both core and Wnt3A exhibit rapid proliferation, enhanced cell cycle progression. Co-expression of core and Wnt3A in HCC cells leads to an accelerated tumor formation in athymic nude mice. Thus, these data strongly suggested that Wnt/β-catenin signaling may be potentiated by core protein and hence play an important role in HCV pathogenesis.2. Human colon cancer cell line HCT116 HEK293 cells For generating adenoviral vector expressing HCV core protein, the full-length of HCV core protein (genotype 1a) was PCR amplified from plasmid H/FL  2 flaks) were infected with an optimal titer of AdWnt3A or AdGFP control. At 15 hr post-infection, the culture medium was changed to serum-free DMEM. Conditioned medium was collected at 48 hr after infection and used immediately.Wnt3A conditioned medium was prepared as described 2 cell culture flasks and transfected with 2 µg per flask of β-catenin/Tcf4-responsive luciferase reporter, pTOP-Luc Renilla luciferase reporter plasmid (Promega) was mixed as an internal control. At 16 hr after transfection, cells were replated to 24-well plates. At 4 hr after replating, cells were infected with AdGFP, Ad-Core, AdWnt3A, AdWnt3A plus Ad-Core or AdWnt3A plus AdGFP. At 24 hr after infection, cells were lysed and subjected to luciferase assays using Promega's Dual Luciferase Kit. Each assay condition was performed in triplicate.Cells were seeded in 25 cmCells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton, and followed by incubating with an anti-β-catenin antibody  overnight. After being washed, cells were incubated with Alexa Fluor 594-labeled secondary antibody (Invitrogen) for 45 min. Cells were counterstained with DAPI to label nuclei. The presence of β-catenin was visualized under a confocal immunofluorescence microscope.Nuclear and cytoplasmic proteins were separated using Pierce's NE-PER Nuclear and Cytoplasmic Extraction Reagent according to the manufacturer's instructions. Whole cell extracts of exponentially growing cells were collected in lysis buffer (Promega) containing the complete cocktail of proteases inhibitors (Roche). Protein concentrations were determined by using the BCA protein assay reagent (Pierce). Approximately 50–70 µg of proteins were resolved on 10% polyacrylamide gels and electrotransferred to PVDF membranes (Millipore). The blots were probed with antibodies against β-catenin , HCV core , Wnt3A , c-Myc , cyclin D1 , GSK3β  and phosphor (Ser9)-GSK3β . Secondary antibodies coupled to HRP were purchased from Jackson ImmunoResearch Laboratories. Proteins of interest were detected with Super Signal West Pico Chemiluminescent substrate Kits (Pierce).c-Myc, cyclin D1, CTGF and WISP2 are listed in Total RNAs were extracted from cultured cells using TRIZOL Reagents (Invitrogen) according to the manufacturer's protocol. Total RNA was used to generate cDNA templates by RT reaction with random hexamer and MMLV RT (Promega). The cDNA products were further diluted 5- to 10-fold and used as qPCR templates. The primer sequences used to detect the expression of HCV core, 2. The absorbance at 490 nm was recorded every 24 hr in triplicate until day 4 by using a microplate reader . Each condition was repeated at least three times.MTS assays were performed with the Cell Titer 96 AQ One Solution Cell Proliferation Assay (Promega). 20 ul MTS reagent was added to each well of the 96 well assay plate and incubated the plate for 2 hr at 37°C in 5% COThe crystal violet staining procedure was carried out as described Cells were prepared for cell cycle analysis as described 6/injection) into the flanks of athymic nude (nu/nu) mice (n = 6 mice per group). At 6 wk after implantation, animals were sacrificed, and tumor masses were retrieved for histological analysis.The care and use of experimental animals was in compliance with the institutional guidelines approved for our study. Athymic nude mice  were used for the studies. Huh7 cells were infected with AdGFP control, Ad-Core, AdGFP plus AdWnt3A, or Ad-Core plus AdWnt3A for 15 hr, and collected for subcutaneous injection  or Ki67  antibodies. Subsequently, the slides were incubated with EnVision System-HRP (DakoCytomation) and visualized using DAB substrate (DakoCytomation).p) of <0.05 was considered statistically significant. Error bars represent SDs of three independent experiments.The data were presented as mean ± SD. Statistical analysis was performed using the SPSS 15.0 software. Differences between groups were compared by the ANOVA test. In all assays, the probability value . Consistent with the immunoblotting results, partially nuclear translocation of β-catenin was observed when cells were infected with Ad-Core but not GFP control . This nuclear translocation effect was further strengthened when core and Wnt3A were co-expressed (n to p), Similar results for nuclear translocation of β-catenin induced by HCV core were obtained in hepatoma cell lines Huh7 cells using immunostaining assay . It is well established that Ser-9 phosphorylation inactivates GSK-3β kinase activity We next investigated the possible mechanism through which core protein induces β-catenin accumulation. Phosphorylation of β-catenin by GSK-3β causes its degradation through the ubiquitin-proteasome system. We examined whether accumulation and stabilization of β-catenin in core-expressing cells was resulted from inactivation of GSK3β. Since β-catenin is mutated and partially localized in the nucleus of HepG2 cells , this cec-Myc, cyclin D1, CTGF and WISP2 are well-known target genes of canonical Wnt signaling p<0.05). Western blot analysis indicated that the protein expression of c-Myc and cyclinD1 were further elevated in these cells than in Wnt3A-treated cells . Quantitative viability analysis confirmed the additive effect of HCV core on Wnt3A-induced growth promotion (p<0.05).We next examined the effect of HCV core protein on biological functions of Wnt/β-catenin signaling. As shown in romotion , p<0.05.p<0.05). Quantitative cell cycle analysis from three batches of experiments showed ectopic expression of core, Wnt3A or core plus Wnt3A significantly promoted G1- to S-phase transition in Huh7 cells, especially at 72 hr and 120 hr post-infection . On the other hand, none of mice injected with GFP control showed tumor formation for up to 10 weeks, indicating that Huh7 cells are not highly tumorigenic under our experimental conditions. Hematoxylin & eosin staining of the retrieved tumor samples revealed that tumor cells with multinuclear and frequent mitosis were surrounded by fibrous connective tissues (a to c). Immunohistochemical (IHC) analysis with a Ki-67 antibody revealed that cell proliferation activities were increased in cells co-infected with Wnt3A plus HCV core. Meanwhile, strong β-catenin protein expression was observed in cytoplasm and whole cells . We also examined the protein expression of Wnt/β-catenin targets c-Myc and cyclin D1 by IHC staining. As shown in j to l) and cyclin D1 (data not shown) after co-expression of Wnt3A and core in hepatoma cells. Taken together, these in vivo results suggest that HCV core protein may potentiate Wnt/β-catenin-induced tumor growth in HCC xenograft model.We further investigated the tumorigenic activity in HCC cells co-infected with core and Wnt3A. Huh7 cells were infected with a comparable titer of AdGFP, Ad-Core, AdWnt3A plus AdGFP or AdWnt3A plus Ad-Core , and theAlthough both virus-induced and immunologically-mediated hepatocytes damage play an important role in pathogenesis, the mechanisms underlying HCV persistence and pathogenesis have been poorly understood. HCV core protein has been reported to play a critical role in HCV-associated liver diseases by activation of several cellular signal transduction, direct interaction with proto-oncogenes c-Myc and cyclin D1. We further demonstrated that cells transduced with core and Wnt3A exhibited increased proliferation, promoted cell cycle progression and accelerated tumor formation in athymic nude mice. Our results are supported by several studies on interaction between virus-encoded proteins and Wnt/β-catenin tumorigenic signaling. For example, HCV NS5A protein was shown to activate β-catenin signaling cascades through increasing the stability of β-catenin In the present study, we sought to investigate whether HCV core protein directly activates Wnt/β-catenin signaling pathway and hence is involved in HCV-induced liver pathogenesis. We found that HCV core protein synergizes Wnt3A-mediated β-catenin-dependent transcriptional activity in hepatocytes and HCC cells. Moreover, co-expression of core and Wnt3A gene induces stabilization and nuclear translocation of β-catenin, contributing to the up-regulation of Wnt-target gene expression, such as The stabilization and accumulation of β-catenin can be achieved by two possible mechanisms. One mechanism is Wnt-dependent stabilization of β-catenin, which was modulated by several components upstream of β-catenin molecule Exogenous core expression was shown to enhance Wnt3A-stimulated HCC tumor growth, possibly by inducing β-catenin accumulation and oncogene overexpression. Interestingly, in xenogarft animal model nuclear translocation of β-catenin in tumor cells was less prominent. There are several possible explanations. First, previous data showed that neoplastic hepatoma cells with nuclear accumulation of β-catenin were restricted to the periphery of tumor nodules or were in small tumor regions detached from large nodules In summary, by co-expressing HCV core protein and Wnt3A in hepatocytes and HCC cell lines, we have found that the HCV core protein can potentiate and synergize with Wnt/β-catenin signaling pathway in promoting cell proliferation and tumor growth. These results strongly suggest that HCV core, possibly through synergizing Wnt-induced stabilization and accumulation of β-catenin, may play an important role in HCV pathogenesis. Nonetheless, the exact role of HCV core in Wnt/β-catenin-induced HCC tumorigenesis remains to be fully investigated. Ultimately, this line of investigation may lead to the development of novel anti-HCV therapies by targeting both HCV itself and the canonical Wnt signaling pathway.Figure S1The recombinant adenovirus effectively expresses core protein in target cells. (A) Effective infection of hepatocytes with Ad-Core. Huh7 or HP14.5 cells were infected with Ad-Core and the infection efficiency was examined under a fluorescence microscope. BF: blank field. Magnification, (400. (B) Ectopic expression of HCV core in Huh7 cells. Huh7 cells were infected with Ad-Core, AdWnt3A or AdGFP control for 36, and protein expression was determined by Western blotting using anti-core antibody (Abcam) (b). Endogenous GAPDH expression was used as a control.(TIF)Click here for additional data file.Figure S2HCV core increases the nuclear translocation of β-catenin induced by Wnt3A. Huh7 cells were infected with AdGFP (panels a to d), Ad-Core (panels e to h), AdWnt3A (panels i to l) or Ad-Core plus AdWnt3A (panels m to p) for 24 hr. Cells were fixed and subjected to immunofluorescence staining as described in (TIF)Click here for additional data file.Figure S3Ectopic expression of HCV core enhances nuclear accumulation of β-catenin in Huh7 and HepG2 cells. Subconfluent hepatoma Huh7 (A) and HepG2 (B) cells were infected with Ad-Core or AdGFP for 24 hr, and stimulated with Wnt3A conditioned medium for 0, 2 and 4 hr. Both cytosolic (C) and nuclear (N) fractions were prepared and subjected to Western blotting analysis with an anti-β-catenin antibody. The subcellular fractions were verified by immunoblotting analysis using either anti-β-tubulin or lamin B1 antibody. WT, wild type; Mut, mutant β-catenin.(TIF)Click here for additional data file.Table S1Cell Cycle (S Phase) analysis.(DOC)Click here for additional data file.


In recent years models have been proposed for the Wnt signaling pathway during the segmentation process in developing embryos. Many of these include negative feedback loops where Axin2 plays a key role. However, Axin2 null mice show no segmentation phenotype. We therefore propose a new model where the negative feedback involves Dkk1 rather than Axin2. We show that this model can exhibit the same type of oscillations as the previous models with Axin2 and as observed in experiments. We show that a spatial Wnt gradient can consistently convert this temporal periodicity into the spatial periodicity of somites, provided the oscillations in new cells arising in the presomitic mesoderm are synchronized with the oscillations of older cells. We further investigate the hypothesis that a change in the Wnt level in the tail bud during the later stages of somitogenesis can lengthen the time period of the oscillations and hence the size and separation of the later somites.The Wnt signaling pathway transducing the stabilization of  A segmented body plan is a fundamental characteristic feature of vertebrates. The process of segmentation is carried out by a combination of changes in gene expression and relative anterior-posterior cell position in the presomitic mesoderm (PSM) In 1976 Cooke and Zeeman In 2003 it was discovered by Aulehla et al. β-catenin interacts with the TCF/LEF-1 DNA-binding proteins to promote transcription of Wnt target genes β-catenin During Wnt signaling β) to the LRP5/6 receptor β (bound to Axin) phosphorylates LRP5/6, which requires Axin β complex to the membrane and the phosphorylation process is thereby amplified It has been proposed that the Wnt signal is transduced through the cell membrane by the binding of Dishevelled (Dsh) to the intracellular domain of the Fz receptor β and then degraded β-catenin, the two kinases GSK3β and casein kinase (CKIα), and the scaffolding proteins Axin and adenomatous polyposis coli (APC). In the destruction complex β-catenin gets phosphorylated and subsequently degraded.At the cell membrane Axin is phosphorylated by GSK3β plays a dual role in controlling the Wnt signal. When the Wnt signal is off then GSK3β phosphorylates β-catenin in the destruction complex and when the signal is on then it phosphorylates Axin at the LRP5/6 receptor. Whether it is the same or distinct Axin-GSK3β complexes that carry out the phosphorylation of β-catenin and Axin is unknown Interestingly enough GSK3α, the scaffolding protein APC, the DNA binding proteins TCF/LEF-1 and the protein Dsh, since their dynamics are not a major part of the negative feedback loop. The dynamics of CK1α and APC are included in the parameters governing the destruction complex, the TCF/LEF-1 dynamics are contained within the transcription of Dkk1, and the dynamics of Dsh are included in the formation of the complex consisting of Axin, GSK3β and LRP5/6 at the cell membrane. C, [GA], G, B, L, Dm, D, [LD], A and [LGA] are the concentrations of the destruction complex, GSK3β-Axin complex, GSK3β, β-catenin , LRP5/6, Dkk1 mRNA, Dkk1 protein, Dkk1-LRP5/6 complex, Axin and LRP5/6-Axin-GSK3β complex. The formation and breaking of a complex X are denoted by B regarding the transcription of mD is associated to the amount of cooperativity between β-catenin and the TCF/LEF-1 complex. For example, no cooperativity would result in a Hill coefficient of one.Our model does not include the dynamics of the kinase CK1β has been shown to be extremely stable G and L. Only constitutive sources, BS and AS, of β-catenin and Axin, respectively, are included in the model, because free (unphosphorylated) β-catenin and Axin is stable The concentration of GSK3Xenopus is proposed and from this article estimates for the dissociation constants for the destruction complex (C) and the [GA] complex, the degradation of β-catenin (α), the total concentration of GSK3β, and the sources of Axin and β-catenin are taken. Dkk1 binds to LRP5/6 with a high affinity; the dissociation constant has been measured to be around The parameters used for our model are listed in The other parameters are estimated to produce oscillations with a period of around 120 min and a very low concentration of total Axin as found by Ref. β-catenin we assume cooperativity between β-catenin and TCF/LEF. The Hill coefficient is set to three. A Hill coefficient of two can produce oscillations, but this requires the affinity of β-catenin to the [GA] complex to be much higher than suggested by the dissociation constant value used in Ref. In the activation of transcription of Dkk1 by β-catenin concentration. This increase will after a while cause an increase in the Dkk1 concentration, which leads to an increase in the [LD] concentration. The high affinity of the [LD] complex leaves little free LPR5/6 behind to form a complex with [GA]. Thus the concentration of the [LGA] complex decreases, leading to an increase in Axin, and the cycle continues. The concentrations of GSK3β and [GA] are mirrors of each other, since a high concentration of [GA] will leave less free GSK3β behind.The parameter values produce oscillations of the involved constituents with a period of around 120 min as seen in β-catenin . For our choice of parameters β-catenin shows an oscillatory behavior with an amplitude of approximately 5nM, which is not significantly low. A different set of parameters could possibly give a smaller amplitude of β-catenin but the general results, presented later, are not significantly altered by this. Even though β-catenin does not oscillate it has been shown that the Notch target gene Nrarp, which stabilizes LEF-1 β-catenin variable in our model can be considered as a coarse-grained variable combining the effects of β-catenin , Nrarp and LEF-1.Ref. KC results in the most drastic changes in amplitude and period of Dkk1  to the LRP5/6 receptor:The reason for using and if we assume steady state for the binding of Wnt to LRP5/6 thenLW] for L in the equations with terms governing the formation of the [LGA] complex, it can be seen that the rate constant for formation of [LGA] will be time dependentWhen substituting  complex needs to be much higher than expected from experiments. The parameters used in this simulation ca be found in (PDF)Click here for additional data file.Figure S2Gaussian profiles of the Wnt gradient. (Top): Gaussian profiles of i denotes the reference state value of i.(PDF)Click here for additional data file.Figure S3Desynchronization of neighboring cells. If the desynchronization between neighboring cells is small (A) then the oscillations of the Dkk1 level at the determination front is almost unaltered. However, if the desynchronization is strong then these oscillations are not appearing.(PDF)Click here for additional data file.Figure S4The Wnt level in the tail bud. (A): When the Wnt level decreases in the tail bud, then the period of the Dkk1 level at the determination front extends. (B): The period is also extended when the Wnt level increases in the tail bud, but the periods drop significantly below the period in the reference state.(PDF)Click here for additional data file.Table S1(PDF)Click here for additional data file.


The Wnt/β-Catenin signaling pathway is central for liver functions and frequently deregulated in hepatocellular carcinoma (HCC). Analysis of the early phenotypes and molecular events following β-Catenin activation is therefore essential for better understanding HCC pathogenesis. The AP-1 transcription factor c-Jun is a putative β-Catenin target gene and promotes hepatocyte survival, proliferation, and liver tumorigenesis, suggesting that c-Jun may be a key target of β-Catenin signaling in the liver.Apc and subsequent β-Catenin activation were analyzed in mice. The contribution of c-Jun to these phenotypes was dissected in double mutant animals lacking both, Apc and c-Jun. β-Catenin was rapidly activated in virtually all Apc mutant hepatocytes while c-Jun was induced only after several days, suggesting that its expression was rather a secondary event following Apc deletion in the liver. Loss of Apc resulted in increased hepatocyte proliferation, hepatomegaly, deregulated protein metabolism, and premature death. Interestingly, additional deletion of c-Jun did not affect hepatocyte proliferation but resulted in increased liver damage and mortality. This phenotype correlated with impaired expression of hepatoprotective genes such as Birc5, Egfr Igf1 and subsequently deregulated Akt signaling.To address this issue, the immediate hepatic phenotypes following deletion of the tumor suppressor These data indicate that c-Jun is not a primary target of β-Catenin signaling in the liver, but rather protects against liver damage, which in turn may promote liver tumorigenesis. Apc results in stabilization and nuclear translocation of β-Catenin, where it cooperates with ternary complex factors (Tcf) to form a ternary complex with DNA responsive elements, thereby regulating expression of its target genes The Wnt/β-Catenin signal transduction cascade is a major regulator of liver development and hepatocyte function Apc or gain of β-Catenin function results in early lethality Apc after birth results in increased hepatocyte proliferation, hepatomegaly and premature mortality within days, possibly due to perturbations in protein and ammonia metabolism Apc acts as tumor suppressor in the liver since its mosaic deletion in only a subset of hepatocytes is sufficient to cause HCC Genetic mouse models have been particularly useful to determine the functions of Apc and β-Catenin in the liver. Constitutive loss of Apc loss are β-Catenin-dependent and rescued upon additional knockout of Ctnnb1 (which encodes β-Catenin) Apc mutant mice are therefore valuable to study the functions of β-Catenin and its target genes in the liver Apc loss in the intestine, but not in the liver, are rescued by additional knockout of c-MycThe immediate phenotypes of Apc loss using double mutant mice lacking both, Apc and c-Jun. Our findings indicate that c-Jun is not essential for hepatocyte proliferation in this setting, but rather protects against liver damage, presumably by interacting with growth factor signaling and subsequent activation of Akt .Several lines of evidence suggest that the transcription factor c-Jun may be an important mediator of β-Catenin functions in the liver. c-Jun is a member of the AP-1 (activator protein 1) complex that contains dimers of either Jun , Fos , Atf (activating transcription factor) or Maf (musculoaponeurotic fibrosarcoma) protein families Apc and c-Jun as well as an inducible Cre recombinase under control of the interferon-responsive Mx1 promoter were generated to analyze the functional impact of c-Jun on the immediate phenotypes of Apc loss in the liver. Mice were injected with double stranded RNA (poly(I·C)) in order to induce Cre-mediated recombination and to generate single Apc knockout (SKO) or Apc/c-Jun double knockout mice (DKO).Compound mutant mice carrying conditional alleles of Apc leads to stabilization and subsequent nuclear translocation of β-Catenin, immunohistochemistry for β-Catenin was performed to functionally assess the Apc recombination efficiency. β-Catenin was predominantly localized on cell membranes in control livers, but translocated to the nucleus in most hepatocytes in SKO and DKO mice as early as 2 days after poly(I·C) injection  injection. Interestingly, median survival was significantly reduced to 8 days in DKO animals, while survival of control animals was not affected , as well as ornithine aminotransferase (Oat) was comparable in SKO and DKO livers as determined by qPCR . These findings indicate that c-Jun protects against liver damage following loss of Apc and that these functions are likely affected by c-Jun gene dosage.c-Jun is a stress-responsive immediate early gene and its delayed expression may reflect increased cell stress occurring at later timepoints following re event . HoweverApc loss . InteresDKO livers, while expression of the apoptosis-related BH3 genes Bim, Bax and Bcl-2 was not affected . However, BiP  expression also occurs during other stress responses such as starvation. In keeping with this notion, hepatic glycogen content was specifically reduced in DKO livers , insulin like growth factor 1 (Igf1) and epidermal growth factor receptor (Egfr) was reduced in affected . Importaatocytes . Igf1 anO livers . ReducedDKO mice . Increasatocytes . HoweverO livers . These fApc mutant mice are an established model to study the immediate liver phenotypes and changes in gene expression following β-Catenin activation Apc and c-Jun.Although Apc may exert functions independent of β-Catenin Apc loss in the liver such as deregulated liver zonation, hepatocyte proliferation and subsequent hepatomegaly. c-Jun has previously been identified as a β-Catenin target gene in the intestine and mediates β-Catenin-dependent intestinal tumorigenesis in ApcMin mice Apc or an activating mutation of Ctnnb1 as well as during colitis-associated colorectal cancer A key finding of our study is the observation that c-Jun is likely not a direct β-Catenin target gene and not an essential mediator of the primary phenotypes of Apc and c-Jun rather resulted in increased liver damage and mortality. It should be noted that these phenotypes may also be influenced by extrahepatic functions of c-Jun, since Mx1Cre-mediated recombination also occurred in extrahepatic tissues such as bone marrow and spleen. It is therefore uncertain whether the increased mortality of DKO mice can be attributed to increased liver damage alone and the extrahepatic functions of c-Jun and causes of death following Apc loss need to be analyzed in more detail in the future. Moreover, it has been shown previously that Mx1Cre-mediated deletion of IKKβ in non-parenchymal liver cells and hepatocytes results in opposite tumorigenesis phenotypes as compared to hepatocyte-specific deletion alone Mx1Cre-mediated deletion of c-Jun, at least during chemically induced liver carcinogenesis and acute T-cell-mediated hepatitis Apc and c-Jun was highly efficient by using Mx1Cre and the hepatic phenotypes observed in these mice closely resembled previous reports of hepatic Apc loss by using different recombination approaches Apc loss.By contrast, hepatic loss of DKO livers correlated with impaired expression of genes implicated in the regulation of hepatocyte survival such as Birc5 and Egfr, which have both been reported as putative direct c-Jun and β-Catenin target genes in other experimental systems Apc and c-Jun resulted in impaired Igf1 expression, which also occurred in livers specifically lacking c-Jun in hepatocytes, suggesting that Igf1 expression is indeed regulated by c-Jun in hepatocytes.The hepatoprotective functions of c-Jun observed here are consistent with the emerging concept that c-Jun promotes hepatocyte survival during diverse stress conditions such as acute hepatitis, hepatic ER stress responses and liver tumorigenesis Apc in the intestine DKO livers, suggesting that c-Jun may directly or indirectly interact with hepatoprotective Akt/mTor signaling in this context. Impaired Akt phosphorylation also correlated with reduced hepatic glycogen content and it is therefore tempting to speculate that c-Jun may be involved in the regulation of metabolic responses in the liver. In addition, Birc5, Egfr, Igf as well as downstream Akt signaling all promote liver tumorigenesis Nutrient availability, insulin and related growth factors regulate cell survival by activation of the serine/threonine kinase Akt, which is a master regulator of cell metabolism, survival, growth and cancer and frequently activated in HCC In conclusion, we provide evidence that c-Jun is not a primary target of β-Catenin signaling in the liver, but rather protects against liver damage in this context. These findings provide a rationale to further explore the interactions of c-Jun with hepatoprotective Akt signaling in metabolic liver disease, hepatocarcinogenesis and tumor cell metabolism.c-Jun or Apc  were crossed with transgenic mice expressing Cre under the control of the interferon-responsive Mx1 promoter Mx1Cre) Apcf/fc-Jun+/+ and Tg(Mx1Cre) Apcf/fc-Junf/f animals were used to generate single (SKO) and double knockout mice (DKO), respectively. Cre-mediated recombination was induced by injection of polyinosine-polycytidylic acid (poly (I·C), 15 µg/g BW, Amersham Biosciences, Piscataway, NJ) and mice were analyzed at the indicated time points. Mice were maintained on a mixed genetic background (C57BL/6×129/Sv) and housed under specific-pathogen free conditions. Littermates not expressing Cre were used as controls. Floxed Apc alleles are considered to have hypomorphic functions Apcf/f c-Junf/f with Apc+/+ c-Junf/f mice revealed no differences in liver size, hepatocyte proliferation or RNA expression of the genes studied here, indicating that the phenotypes of the controls used here was not substantially affected by the floxed Apc alleles  c-Junf/f, c-JunΔli) were used Mice with conditional alleles for  alleles . For somin situ cell death detection kit .For histology, livers were fixed in 3.7% neutral buffered formaldehyde at 4°C overnight and embedded in paraffin. Immunohistochemistry was performed using the Envision kit  and antibodies for c-Jun , β-Catenin , Glutamine synthetase , Ki67  and BiP . Slides were counterstained with Hematoxylin . H&E stainings were performed according to standard protocols. Microscopy was performed on a Zeiss Observer Z1 with SPOT Imaging software. The percentage of hepatocytes expressing c-Jun, β-Catenin or Ki67 was determined by counting hepatocyte nuclei within 3 random 400× magnification fields per mouse (n ≥ 6 livers per genotype). TUNEL assays were performed using the Hepatocyte damage was determined by the level of serum transaminases and serum glucose concentrations were analyzed using semi-automated clinical routine methods. Liver glycogen concentrations were analyzed using a glycogen assay kit  according to the manufacturer's protocol.Total liver lysates were analyzed by immunoblot using antibodies for β-Actin (Sigma), insulin receptor , BiP , p-Akt  and Akt . Serum insulin concentrations were determined using the insulin (rat) ultrasensitive ELISA kit with 75% mouse sensitivity .Hprt. Primer sequences are available upon request.Upon isolation of total liver RNA using Qiazol , cDNA synthesis was performed using the first strand cDNA synthesis kit . qPCR was performed with SYBR Green  on a 480 Lightcycler (Roche) and the expression levels of transcripts were calculated with the comparative CT method. The individual RNA levels were normalized for t test. Statistical analysis of mouse survival was determined by log rank test.Data in bar graphs represent mean ± S.D. as indicated. Statistical analysis was performed using the nondirectional two-tailed Student's Figure S1Expression of the indicated genes was analyzed in control livers and livers from mice specifically lacking c-Jun in hepatocytes (c-JunΔli). (n = 3 livers/genotype); *, P≤0.05; §, P≤0.01.(TIF)Click here for additional data file.Figure S2Hypomorphic floxed Apcalleles do not affect liver size, hepatocyte proliferation and hepatic gene expression. (A) Liver weight of mice with the indicated genotypes was determined and is given as liver/body weight ratio in [%]. (n>4 mice/genotype). (B) The percentage of Ki67 positive hepatocytes is shown in [%] (n>4 livers/genotype). (C) Hepatic expression of the indicated genes was determined by qPCR and is given as relative expression compared to control livers (n>3 livers/genotype).(TIF)Click here for additional data file.