TLR4 Signaling via NANOG Cooperates With STAT3 to Activate Twist1 and Promote Formation of Tumor-Initiating Stem-Like Cells in Livers of Mice.

BACKGROUND & AIMS: Obesity and alcohol consumption contribute to steatohepatitis, which increases the risk for hepatitis C virus (HCV)-associated hepatocellular carcinomas (HCCs). Mouse hepatocytes that express HCV-NS5A in liver up-regulate the expression of Toll-like receptor 4 (TLR4), and develop liver tumors containing tumor-initiating stem-like cells (TICs) that express NANOG. We investigated whether the TLR4 signals to NANOG to promote the development of TICs and tumorigenesis in mice placed on a Western diet high in cholesterol and saturated fat (HCFD). METHODS: We expressed HCV-NS5A from a transgene (NS5A Tg) in Tlr4-/- (C57Bl6/10ScN), and wild-type control mice. Mice were fed a HCFD for 12 months. TICs were identified and isolated based on being CD133+, CD49f+, and CD45-. We obtained 142 paraffin-embedded sections of different stage HCCs and adjacent nontumor areas from the same patients, and performed gene expression, immunofluorescence, and immunohistochemical analyses. RESULTS: A higher proportion of NS5A Tg mice developed liver tumors (39%) than mice that did not express HCV NS5A after the HCFD (6%); only 9% of Tlr4-/- NS5A Tg mice fed HCFD developed liver tumors. Livers from NS5A Tg mice fed the HCFD had increased levels of TLR4, NANOG, phosphorylated signal transducer and activator of transcription (pSTAT3), and TWIST1 proteins, and increases in Tlr4, Nanog, Stat3, and Twist1 messenger RNAs. In TICs from NS5A Tg mice, NANOG and pSTAT3 directly interact to activate expression of Twist1. Levels of TLR4, NANOG, pSTAT3, and TWIST were increased in HCC compared with nontumor tissues from patients. CONCLUSIONS: HCFD and HCV-NS5A together stimulated TLR4-NANOG and the leptin receptor (OB-R)-pSTAT3 signaling pathways, resulting in liver tumorigenesis through an exaggerated mesenchymal phenotype with prominent Twist1-expressing TICs.

Enhanced treatment of breast cancer brain metastases with oncolytic virus expressing anti-CD47 antibody and temozolomide.

Limited therapeutic options are available for patients with breast cancer brain metastases (BCBM), and thus there is an urgent need for novel treatment approaches. We previously engineered an effective oncolytic herpes simplex virus 1 (oHSV) expressing a full-length anti-CD47 monoclonal antibody (mAb) with a human IgG1 scaffold (OV-αCD47-G1) that was used to treat both ovarian cancer and glioblastoma. Here, we demonstrate that the combination of OV-αCD47-G1 and temozolomide (TMZ) improve outcomes in preclinical models of BCBM. The combination of TMZ with OV-αCD47-G1 synergistically increased macrophage phagocytosis against breast tumor cells and led to greater activation of NK cell cytotoxicity. In addition, the combination of OV-αCD47-G1 with TMZ significantly prolonged the survival of tumor-bearing mice when compared with TMZ or OV-αCD47-G1 alone. Combination treatment with the mouse counterpart of OV-αCD47-G1, termed OV-A4-IgG2b, also enhanced mouse macrophage phagocytosis, NK cell cytotoxicity, and survival in an immunocompetent model of mice bearing BCBM compared with TMZ or OV-A4-IgG2b alone. Collectively, these results suggest that OV-αCD47-G1 combined with TMZ should be explored in patients with BCBM.

Cancer stem cells generated by alcohol, diabetes, and hepatitis C virus.

Cancer stem cells (tumor-initiating stem-like cells: TISCs) are resistant to chemotherapy and are associated with metastatic hepatocellular carcinoma (HCC), which is commonly observed in hepatitis C virus (HCV)-infected patients with obesity or alcohol abuse. However, it is unknown whether the TLR4-NANOG pathway serves as a universal oncogenic signaling in the genesis of TISCs and HCC. We aimed to determine whether Tlr4 is a putative proto-oncogene for TISCs in liver oncogenesis due to different etiologies and how Tlr4 is regulated at the transcriptional and epigenetic levels. CD133+/CD49f+ TISCs were isolated using FACS from HCC developed in HCV Core Tg mice fed alcohol, diethylnitrosamine-treated mice, and alcoholic patients with or without HCV infection. CD133+/CD49f+ cells isolated from the animal models and patients are tumorigenic both in vitro and in a xenograft model, and Tlr4 or Nanog silencing with shRNA attenuates their tumor initiating property. Functional oncogene screening of a cDNA library identified the organ size control pathway targets Yap1 and AKT activator Igf2bp3 as NANOG-dependent genes that inhibit transforming growth factor-ß signaling in TISCs. Tlr4 expression is higher in TISCs compared with CD133-/CD49f+ cells. Taken together, Tlr4 may be a universal proto-oncogene responsible for the genesis of TLR4-NANOG dependent TISCs, and this pathway serves as a novel therapeutic target for HCC.

c-Jun mediates hepatitis C virus hepatocarcinogenesis through signal transducer and activator of transcription 3 and nitric oxide-dependent impairment of oxidative DNA repair.

UNLABELLED: Hepatocellular carcinoma (HCC) occurs in a significant number of patients with hepatitis C virus (HCV) infection. HCV causes double-strand DNA breaks and enhances the mutation frequency of proto-oncogenes and tumor suppressors. However, the underlying mechanisms for these oncogenic events are still elusive. Here, we studied the role of c-Jun, signal transducer and activator of transcription 3 (STAT3), and nitric oxide (NO) in spontaneous and diethylnitrosamine (DEN)-initiated and/or phenobarbital (Pb)-promoted HCC development using HCV core transgenic (Tg) mice. The viral core protein induces hepatocarcinogenesis induction as a tumor initiator under promotion by Pb treatment alone. Conditional knockout of c-jun and stat3 in hepatocytes achieves a nearly complete, additive effect on prevention of core-induced spontaneous HCC or core-enhanced HCC incidence caused by DEN/Pb. Core protein induces hepatocyte proliferation and the expression of inflammatory cytokines (interleukin-6, tumor necrosis factor-alpha, interleukin-1) and inducible NO synthase (iNOS); the former is dependent on c-Jun and STAT3, and the latter on c-Jun. Oxidative DNA damage repair activity is impaired by the HCV core protein due to reduced DNA glycosylase activity for the excision of 8-oxo-2'-deoxyguanosine. This impairment is abrogated by iNOS inhibition or c-Jun deficiency, but aggravated by the NO donor or iNOS-inducing cytokines. The core protein also suppresses apoptosis mediated by Fas ligand because of c-Jun-dependent Fas down-regulation. CONCLUSION: These results indicate that the HCV core protein potentiates chemically induced HCC through c-Jun and STAT3 activation, which in turn, enhances cell proliferation, suppresses apoptosis, and impairs oxidative DNA damage repair, leading to hepatocellular transformation.

Cullin 4B regulates cell survival and apoptosis in clear cell renal cell carcinoma as a target of microRNA-217.

Cullin 4B (CUL4B) was reported to be closely related to the progression of some tumors, but its function in clear cell renal cell carcinoma (ccRCC) has not been reported. Our present study found CUL4B was upregulated in ccRCC, and CUL4B knockdown markedly inhibited ccRCC cell growth and induced apoptosis. In addition, CUL4B knockdown markedly inhibited antiapoptotic proteins' expression in ccRCC cells, including Mcl-1 and Bcl-2, and silenced CUL4B also induced the cleavages of PARP, an important index of apoptosis. We also confirmed microRNA-217 (miR-217) was downregulated in ccRCC tumor tissues, and negatively correlated with CUL4B expression. Further investigations revealed miR-217 targeted CUL4B and markedly inhibited its expression in ccRCC cells. In addition, overexpression of miR-217 by mimics significantly suppressed ccRCC cell growth. In contrast, enforced expression of CUL4B significantly abolished miR-217-induced cell survival inhibition in ccRCC cells. In conclusion, our present results suggested targeting miR-217-CUL4B axis would be a promising strategy for ccRCC treatment.

Hepatitis C virus causes uncoupling of mitotic checkpoint and chromosomal polyploidy through the Rb pathway.

Hepatitis C virus (HCV) infection is associated with the development of hepatocellular carcinoma and probably also non-Hodgkin's B-cell lymphoma. The molecular mechanisms of HCV-associated carcinogenesis are unknown. Here we demonstrated that peripheral blood mononuclear cells obtained from hepatitis C patients and hepatocytes infected with HCV in vitro showed frequent chromosomal polyploidy. HCV infection or the expression of viral core protein alone in hepatocyte culture or transgenic mice inhibited mitotic spindle checkpoint function because of reduced Rb transcription and enhanced E2F-1 and Mad2 expression. The silencing of E2F-1 by RNA interference technology restored the function of mitotic checkpoint in core-expressing cells. Taken together, these data suggest that HCV infection may inhibit the mitotic checkpoint to induce polyploidy, which likely contributes to neoplastic transformation.

Contamination of soils with organochlorine pesticides in urban parks in Beijing, China.

Urban parks are an integral component of healthy urban living. Since they are frequently visited, an understanding of the environmental quality of these urban facilities is crucial. Here, a study was conducted on the contamination of soils in the parks of Beijing. Organochlorine pesticides (OCPs), which have the potential to cause endocrine disturbances, were considered study objectives. Hexachlorocyclohexanes (HCHs) were found at concentrations of 0.2490-197.0 ng g(-1) and dichlorodiphenyltrichloroethanes (DDTs) were found at concentrations of 5.942-1039 ng g(-1) in the soils investigated. The preliminary pollution assessment indicated that DDTs have caused high pollution levels in the soils of some parks. Analysis of the sources of contamination showed that HCHs in the soils were derived from an old mixed source of technical HCHs and lindane and that DDTs, which were suspected to have recent application to the soils at some sites, were derived mainly from a mixture of technical DDTs and dicofol containing DDT impurities. An independent sample t-test proved that pesticides containing DDTs had been used in large amounts in the soils of parks before 1983 (p<0.05) and that the levels of DDTs in the soils of parks administered by the Beijing municipal government were significantly higher than the levels in those administered by the district government (p<0.05). However, the main difference in this situation needs to be further studied. This study suggested that open spaces like urban parks were not as sound as was expected and that there was potential for exposure of visitors/workers in the parks to organochlorine pesticides.

Diversity of fish sound types in the Pearl River Estuary, China.

BACKGROUND: Repetitive species-specific sound enables the identification of the presence and behavior of soniferous species by acoustic means. Passive acoustic monitoring has been widely applied to monitor the spatial and temporal occurrence and behavior of calling species. METHODS: Underwater biological sounds in the Pearl River Estuary, China, were collected using passive acoustic monitoring, with special attention paid to fish sounds. A total of 1,408 suspected fish calls comprising 18,942 pulses were qualitatively analyzed using a customized acoustic analysis routine. RESULTS: We identified a diversity of 66 types of fish sounds. In addition to single pulse, the sounds tended to have a pulse train structure. The pulses were characterized by an approximate 8 ms duration, with a peak frequency from 500 to 2,600 Hz and a majority of the energy below 4,000 Hz. The median inter-pulsepeak interval (IPPI) of most call types was 9 or 10 ms. Most call types with median IPPIs of 9 ms and 10 ms were observed at times that were exclusive from each other, suggesting that they might be produced by different species. According to the literature, the two section signal types of 1 + 1 and 1 + N10 might belong to big-snout croaker (Johnius macrorhynus), and 1 + N19 might be produced by Belanger's croaker (J. belangerii). DISCUSSION: Categorization of the baseline ambient biological sound is an important first step in mapping the spatial and temporal patterns of soniferous fishes. The next step is the identification of the species producing each sound. The distribution pattern of soniferous fishes will be helpful for the protection and management of local fishery resources and in marine environmental impact assessment. Since the local vulnerable Indo-Pacific humpback dolphin (Sousa chinensis) mainly preys on soniferous fishes, the fine-scale distribution pattern of soniferous fishes can aid in the conservation of this species. Additionally, prey and predator relationships can be observed when a database of species-identified sounds is completed.

Apparent source levels and active communication space of whistles of free-ranging Indo-Pacific humpback dolphins (Sousa chinensis) in the Pearl River Estuary and Beibu Gulf, China.

Background. Knowledge of species-specific vocalization characteristics and their associated active communication space, the effective range over which a communication signal can be detected by a conspecific, is critical for understanding the impacts of underwater acoustic pollution, as well as other threats. Methods. We used a two-dimensional cross-shaped hydrophone array system to record the whistles of free-ranging Indo-Pacific humpback dolphins (Sousa chinensis) in shallow-water environments of the Pearl River Estuary (PRE) and Beibu Gulf (BG), China. Using hyperbolic position fixing, which exploits time differences of arrival of a signal between pairs of hydrophone receivers, we obtained source location estimates for whistles with good signal-to-noise ratio (SNR ≥10 dB) and not polluted by other sounds and back-calculated their apparent source levels (ASL). Combining with the masking levels (including simultaneous noise levels, masking tonal threshold, and the Sousa auditory threshold) and the custom made site-specific sound propagation models, we further estimated their active communication space (ACS). Results. Humpback dolphins produced whistles with average root-mean-square ASL of 138.5 ± 6.8 (mean ± standard deviation) and 137.2 ± 7.0 dB re 1 µPa in PRE (N = 33) and BG (N = 209), respectively. We found statistically significant differences in ASLs among different whistle contour types. The mean and maximum ACS of whistles were estimated to be 14.7 ± 2.6 (median ± quartile deviation) and 17.1± 3.5 m in PRE, and 34.2 ± 9.5 and 43.5 ± 12.2 m in BG. Using just the auditory threshold as the masking level produced the mean and maximum ACSat of 24.3 ± 4.8 and 35.7 ± 4.6 m for PRE, and 60.7 ± 18.1 and 74.3 ± 25.3 m for BG. The small ACSs were due to the high ambient noise level. Significant differences in ACSs were also observed among different whistle contour types. Discussion. Besides shedding some light for evaluating appropriate noise exposure levels and information for the regulation of underwater acoustic pollution, these baseline data can also be used for aiding the passive acoustic monitoring of dolphin populations, defining the boundaries of separate groups in a more biologically meaningful way during field surveys, and guiding the appropriate approach distance for local dolphin-watching boats and research boat during focal group following.

Passive Acoustic Monitoring the Diel, Lunar, Seasonal and Tidal Patterns in the Biosonar Activity of the Indo-Pacific Humpback Dolphins (Sousa chinensis) in the Pearl River Estuary, China.

A growing demand for sustainable energy has led to an increase in construction of offshore windfarms. Guishan windmill farm will be constructed in the Pearl River Estuary, China, which sustains the world's largest known population of Indo-Pacific humpback dolphins (Sousa chinensis). Dolphin conservation is an urgent issue in this region. By using passive acoustic monitoring, a baseline distribution of data on this species in the Pearl River Estuary during pre-construction period had been collected. Dolphin biosonar detection and its diel, lunar, seasonal and tidal patterns were examined using a Generalized Linear Model. Significant higher echolocation detections at night than during the day, in winter-spring than in summer-autumn, at high tide than at flood tide were recognized. Significant higher echolocation detections during the new moon were recognized at night time. The diel, lunar and seasonal patterns for the echolocation encounter duration also significantly varied. These patterns could be due to the spatial-temporal variability of dolphin prey and illumination conditions. The baseline information will be useful for driving further effective action on the conservation of this species and in facilitating later assessments of the effects of the offshore windfarm on the dolphins by comparing the baseline to post construction and post mitigation efforts.

Development of a scalable suspension culture for cardiac differentiation from human pluripotent stem cells.

To meet the need of a large quantity of hPSC-derived cardiomyocytes (CM) for pre-clinical and clinical studies, a robust and scalable differentiation system for CM production is essential. With a human pluripotent stem cells (hPSC) aggregate suspension culture system we established previously, we developed a matrix-free, scalable, and GMP-compliant process for directing hPSC differentiation to CM in suspension culture by modulating Wnt pathways with small molecules. By optimizing critical process parameters including: cell aggregate size, small molecule concentrations, induction timing, and agitation rate, we were able to consistently differentiate hPSCs to >90% CM purity with an average yield of 1.5 to 2×10(9) CM/L at scales up to 1L spinner flasks. CM generated from the suspension culture displayed typical genetic, morphological, and electrophysiological cardiac cell characteristics. This suspension culture system allows seamless transition from hPSC expansion to CM differentiation in a continuous suspension culture. It not only provides a cost and labor effective scalable process for large scale CM production, but also provides a bioreactor prototype for automation of cell manufacturing, which will accelerate the advance of hPSC research towards therapeutic applications.

[Superposition impact character of air pollution from decentralization docks in a freshwater port].

Air pollution from freshwater port is mainly caused by dust pollution, including material loading and unloading dust, road dust, and wind erosion dust from stockpile, bare soil. The dust pollution from a single dock characterized in obvious difference with air pollution from multiple scattered docks. Jining Port of Shandong Province was selected as a case study to get superposition impact contribution of air pollution for regional air environment from multiple scattered docks and to provide technical support for system evaluation of port air pollution. The results indicate that (1) the air pollution from freshwater port occupies a low proportion of pollution impact on regional environmental quality because the port is consisted of serveral small scattered docks; (2) however, the geometric center of the region distributed by docks is severely affected with the most superposition of the air pollution; and (3) the ADMS model is helpful to attain an effective and integrated assessment to predict a superposition impact of multiple non-point pollution sources when the differences of high-altitude weather conditions was not considered on a large scale.

Reciprocal regulation by TLR4 and TGF-ß in tumor-initiating stem-like cells.

Tumor-initiating stem-like cells (TICs) are resistant to chemotherapy and associated with hepatocellular carcinoma (HCC) caused by HCV and/or alcohol-related chronic liver injury. Using HCV Tg mouse models and patients with HCC, we isolated CD133(+) TICs and identified the pluripotency marker NANOG as a direct target of TLR4, which drives the tumor-initiating activity of TICs. These TLR4/NANOG-dependent TICs were defective in the TGF-ß tumor suppressor pathway. Functional oncogene screening of a TIC cDNA library identified Yap1 and Igf2bp3 as NANOG-dependent genes that inactivate TGF-ß signaling. Mechanistically, we determined that YAP1 mediates cytoplasmic retention of phosphorylated SMAD3 and suppresses SMAD3 phosphorylation/activation by the IGF2BP3/AKT/mTOR pathway. Silencing of both YAP1 and IGF2BP3 restored TGF-ß signaling, inhibited pluripotency genes and tumorigenesis, and abrogated chemoresistance of TICs. Mice with defective TGF-ß signaling (Spnb2(+/-) mice) exhibited enhanced liver TLR4 expression and developed HCC in a TLR4-dependent manner. Taken together, these results suggest that the activated TLR4/NANOG oncogenic pathway is linked to suppression of cytostatic TGF-ß signaling and could potentially serve as a therapeutic target for HCV-related HCC.

[Impact of water pollution risk in water transfer project based on fault tree analysis].

The methods to assess water pollution risk for medium water transfer are gradually being explored. The event-nature-proportion method was developed to evaluate the probability of the single event. Fault tree analysis on the basis of calculation on single event was employed to evaluate the extent of whole water pollution risk for the channel water body. The result indicates, that the risk of pollutants from towns and villages along the line of water transfer project to the channel water body is at high level with the probability of 0.373, which will increase pollution to the channel water body at the rate of 64.53 mg/L COD, 4.57 mg/L NH4(+) -N and 0.066 mg/L volatilization hydroxybenzene, respectively. The measurement of fault probability on the basis of proportion method is proved to be useful in assessing water pollution risk under much uncertainty.

[Risk assessment and safety evaluation using system normative indexes integration method for non-point source pollution on watershed scale].

Decision-makers take non-point source pollution under control as well as possible results from enough information of risk trend of nonpoint source pollution on watershed scale. System normative indexes integration evaluation method about system risk trend was developed when focusing on that the probability values of some elements attributing to some trend of the system were more than one, and that the system evaluation needed a formula from the system structure. On the basis of analysis on aspects and characteristics of the system risk normalization, a new valuation method, the relationship between the normalization values of the system and the factors was established. The Lugu Lake Watershed in Southwest China was selected as study area to assess the risk of non-point source loss to surface water using this method. The results indicate that 1) the wholly risk of non-point source loss to surface water in this watershed is in a high level; 2) the system indexes integration evaluation method is an universal method to evaluate a quality or a trend of any system and shows a great power in comparing several systems; 3) the method is helpful to attain an effective and integrated assessment on a system when it is combined with other methods.

Characterization of acetanilide herbicides degrading bacteria isolated from tea garden soil.

Three different green manures were added to the tea garden soils separately and incubated for 40 days. After, incubation, acetanilide herbicides alachlor and metolachlor were spiked into the soils, separately, followed by the isolation of bacteria in each soil at designed intervals. Several bacterial strains were isolated from the soils and identified as Bacillus silvestris, B. niacini, B. pseudomycoides, B. cereus, B. thuringiensis, B. simplex, B. megaterium, and two other Bacillus sp. (Met1 and Met2). Three unique strains with different morphologies were chosen for further investigation. They were B. megaterium, B. niacini, and B. silvestris. The isolated herbicide-degrading bacteria showed optimal performance among three incubation temperatures of 30 degrees C and the best activity in the 10 to 50 microg/ml concentration of the herbicide. Each bacterial strain was able to degrade more than one kind of test herbicides. After incubation for 119 days, B. cereus showed the highest activity to degrade alachlor and propachlor, and B. thuringiensis to degrade metolachlor.