LC/MS-Based Global Metabolomic Identification of Serum Biomarkers Differentiating Hepatocellular Carcinoma from Chronic Hepatitis B and Liver Cirrhosis.

Chronic hepatitis B virus (CHB) infection is one of the primary risk factors associated with the development of hepatocellular carcinoma (HCC). Despite having been extensively studied, diagnosing early-stage HCC remains challenging, and diagnosed patients have a poor (3-5%) survival rate. Identifying new approaches to detect changes in the serum metabolic profiles of patients with CHB and liver cirrhosis (LC) may provide a valuable approach to better detect HCC at an early stage when it is still amenable to treatment, thereby improving patient prognosis and survival. In the present study, we, therefore, employed a liquid chromatography-mass spectrometry (LC-MS)-based approach to evaluate the serum metabolic profiles of 30 CHB patients, 29 LC patients, and 30 HCC patients. We then employed appropriate statistical methods to identify those metabolites that were best able to distinguish HCC cases from LC and CHB controls. A mass-based database was then used to putatively identify these metabolites. We then confirmed the identities of a subset of these metabolites through comparisons with the MS/MS fragmentation patterns and retention times of reference standards. The serum samples were then reanalyzed to quantify the levels of these selected metabolites and of other metabolites that have previously been identified as potential HCC biomarkers. Through this approach, we observed clear differences in the metabolite profiles of the CHB, LC, and HCC patient groups in both positive- and negative-ion modes. We found that the levels of taurodeoxy cholic acid (TCA) and 1,2-diacyl-3-ß-d-galactosyl-sn-glycerol rose with the progression from CHB to LC to HCC, whereas levels of 5-hydroxy-6E,8Z,11Z,14Z,17Z-eicosapentaenoic acid, and glycyrrhizic acid were gradually reduced with liver disease progression in these groups. The ROC analysis showed that taurodeoxy cholic acid (TCA), 1,2-diacyl-3-ß-d-galactosyl-sn-glycerol, 5-hydroxy-6E,8Z,11Z,14Z,17Z-eicosapentaenoic acid, and glycyrrhizic acid had a diagnosis performance with liver disease progression. These four metabolites have a significant correlation with alpha fetal protein (AFP) level and age. Our results highlight novel metabolic biomarkers that have the potential to be used for differentiating between CHB, LC, and HCC patients, thereby facilitating the identification and treatment of patients with early-stage HCC.

MiR-218 suppresses metastasis and invasion of endometrial cancer via negatively regulating ADD2.

OBJECTIVE: Endometrial cancer (EC) is one of the three most common gynecological cancers. Due to the lack of effective treatment for EC patients in an advanced stage, the mortality rate of EC is increasing rapidly. Hence, it is essential to seek for novel molecular therapeutic targets and biomarkers for EC. The aim of this study was to explore the role of miR-218 in the occurrence and development of EC and to investigate the possible underlying mechanism. PATIENTS AND METHODS: The expression level of miR-218 in EC tissues and cell lines was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Wound healing assay and Matrigel assay were performed to determine the migration and invasion abilities of EC cells. Meanwhile, the potential targets of miR-218 were predicted by bioinformatics analysis and confirmed by Luciferase reporter gene assay. In addition, the protein expression level of Adducin 2 (ADD2) was assessed by Western blotting analysis. RESULTS: QRT-PCR results revealed that miR-218 was significantly downregulated in EC tissues and cell lines. Wound healing assay and Matrigel assay demonstrated that miR-218 suppressed the migration and invasion abilities of EC cells. Online prediction databases predicted that ADD2 was a direct target of miR-218, which was verified by Luciferase reporter gene assay. Rescue experiments further validated that miR-218 could serve as a carcinoma suppressor by negatively regulating ADD2 expression in EC. CONCLUSIONS: In the present study, we elucidated that miR-218 served as a tumor suppressor in EC by negatively regulating ADD2. This might bring a novel insight into new molecular therapeutic targets and biomarkers for EC.

Maghemite nanoparticles coated with human serum albumin: combining targeting by the iron-acquisition pathway and potential in photothermal therapies.

Human serum albumin (HSA), the most abundant plasma protein in human blood, is a natural transport vehicle with multiple ligand binding sites. It, therefore, constitutes an attractive candidate for drug delivery. Targeting may occur via the most known interaction of the protein with the neonatal Fc receptor (FcRn). Here, we investigate another HSA delivery path, involving the transferrin receptor, and we elaborate a maghemite-HSA nanohybrid, opening up new opportunities for medical applications. Fluorescence spectrophotometric titration and size-exclusion chromatography were used to substantiate, in cell-free assays, an interaction between HSA and the transferrin receptor R1. This occurs with a dissociation constant, KD of 6.7 nM. This interaction was confirmed in HeLa cell culture where, by confocal microscopy, rhodamine-labeled HSA is shown to be internalized. HSA was then covalently conjugated onto maghemite nanoparticles (NPs) to give a NP-HSA nanohybrid. The therapeutic potential of this hybrid was demonstrated through its heating capacity in magnetic hyperthermia (MH) and near-infrared (NIR) photothermia (PT). In particular, the Specific Absorption Rate (SAR) in the PT Therapy (PTT) mode, using a 808 nm NIR-LASER (1 W cm-2) and at iron concentration as low as 2.5 mM, was found to be very high, equal to 1870 W g-1 with a temperature increment of 9.2 °C. The nanohybrids incubated with HeLa cells were mainly localized at the cell surface. When the PTT mode was applied under the same conditions as in vitro, mortality was higher in HeLa cells than in fibroblasts (non-malignant cells). Cytotoxicity was checked in both cell lines without the PTT mode; the nanohybrids do not seem to affect cell viability. These results make the nanohybrids very promising agents for NIR-PT and for targeting in cancer therapy, since non-malignant cells were not damaged.

DIFFERENTIAL PATHWAY DEPENDENCY DISCOVERY ASSOCIATED WITH DRUG RESPONSE ACROSS CANCER CELL LINES.

The effort to personalize treatment plans for cancer patients involves the identification of drug treatments that can effectively target the disease while minimizing the likelihood of adverse reactions. In this study, the gene-expression profile of 810 cancer cell lines and their response data to 368 small molecules from the Cancer Therapeutics Research Portal (CTRP) are analyzed to identify pathways with significant rewiring between genes, or differential gene dependency, between sensitive and non-sensitive cell lines. Identified pathways and their corresponding differential dependency networks are further analyzed to discover essentiality and specificity mediators of cell line response to drugs/compounds. For analysis we use the previously published method EDDY (Evaluation of Differential DependencY). EDDY first constructs likelihood distributions of gene-dependency networks, aided by known genegene interaction, for two given conditions, for example, sensitive cell lines vs. non-sensitive cell lines. These sets of networks yield a divergence value between two distributions of network likelihoods that can be assessed for significance using permutation tests. Resulting differential dependency networks are then further analyzed to identify genes, termed mediators, which may play important roles in biological signaling in certain cell lines that are sensitive or non-sensitive to the drugs. Establishing statistical correspondence between compounds and mediators can improve understanding of known gene dependencies associated with drug response while also discovering new dependencies. Millions of compute hours resulted in thousands of these statistical discoveries. EDDY identified 8,811 statistically significant pathways leading to 26,822 compound-pathway-mediator triplets. By incorporating STITCH and STRING databases, we could construct evidence networks for 14,415 compound-pathway-mediator triplets for support. The results of this analysis are presented in a searchable website to aid researchers in studying potential molecular mechanisms underlying cells' drug response as well as in designing experiments for the purpose of personalized treatment regimens.

[The role of hexokinase 2 in the metastasis of hepatocellular carcinoma cells].

Objective: To investigate the regulatory role of HK2 in the metastasis of hepatocellular carcinoma (HCC). Methods: The protein expressions of HK2 in 73 HCC tumor tissues and paired adjacent non-tumor tissues were evaluated by using immunohistochemical analysis. The scratch wound healing assay and Transwell assay had been used to analyze the migration and invasion of HCC cells with HK2 knockdown. Expressions of epithelial-to-mesenchymal transition (EMT)markers, such as E-cadherin, ZO-1, N-cadherin and vimentin, in HCC cells with HK2 knockdown were determined by qRT-PCR and Western blot analysis. Results: The expression levels of HK2 in tumor tissues and adjacent non-tumor tissues were 5.39±3.40 and 2.16±1.55, respectively. The protein expression of HK2 was significantly higher in tumor tissues compared with adjacent non-tumor tissues of HCC (P<0.05). Knockdown of HK2 in HCC cells decreased the cell motility from 1.00±0.54 to 0.56±0.09 (P<0.05), andknockdown of HK2 in HCC cells decreased the number of invaded cells form 345±42 to 215±34 (P<0.05). The expression of epithelial markers ZO-1 and E-cadherin were up-regulated, while mesenchymal markers vimentin and N-cadherin were down-regulated in HCC cells when HK2 was knockeddown. Conclusions: HK2 is up-regulated in HCC and promotes cell motility by stimulating EMT.

Interactions between fibroin and sericin proteins from Antheraea pernyi and Bombyx mori silk fibers.

Silkworm silk fibers are core-shell composites of fibroin and sericin proteins. Studying the interactions between fibroin and sericin is essential for understanding the properties of these composites. It is observed that compared to the domestic silk cocoon Bombyx mori (B. mori), the adhesion between fibroin and sericin from the wild silk cocoon, Antheraea pernyi (A. pernyi), is significantly stronger with a higher degree of heterogeneity. The adsorption of A. pernyi sericin on its fibroin is almost twice the value for B. mori sericin on fibroin, both showing a monolayer Langmuir adsorption. (1)H NMR and FTIR studies demonstrate on a molecular level the stronger interactions and the more intensive complex formation between A. pernyi fibroin and sericin, facilitated by the hydrogen bonding between glycine and serine. The findings of this study may help the design of composites with superior interfacial adhesion between different components.

Integrin α11ß1 regulates cancer stromal stiffness and promotes tumorigenicity and metastasis in non-small cell lung cancer.

Integrin α11ß1 is a stromal cell-specific receptor for fibrillar collagens and is overexpressed in carcinoma-associated fibroblasts (CAFs). We have investigated its direct role in cancer progression by generating severe combined immune deficient (SCID) mice deficient in integrin α11 (α11) expression. The growth of A549 lung adenocarcinoma cells and two patient-derived non-small cell lung carcinoma (NSCLC) xenografts in these α11 knockout (α11(-/-)) mice was significantly impeded, as compared with wild-type (α11(+/+)) SCID mice. Orthotopic implantation of a spontaneously metastatic NCI-H460SM cell line into the lungs of α11(-/-) and α11(+/+) mice showed significant reduction in the metastatic potential of these cells in the α11(-/-) mice. We identified that collagen cross-linking is associated with stromal α11 expression, and the loss of tumor stromal α11 expression was correlated with decreased collagen reorganization and stiffness. This study shows the role of integrin α11ß1, a receptor for fibrillar collagen in differentiation of fibroblasts into CAFs. Furthermore, our data support an important role for α11 signaling pathway in CAFs, promoting tumor growth and metastatic potential of NSCLC cells and being closely associated with collagen cross-linking and the organization and stiffness of fibrillar collagen matrices.

Concentrations and distributions of 18 organochlorine pesticides listed in the Stockholm Convention in surface sediments from the Liaohe River basin, China.

Organochlorine pesticides (OCPs) were analyzed in 26 surface sediment samples from the Liaohe River basin, and the distributions of and potential environmental risks posed by OCPs in the basin were evaluated. Eighteen OCPs listed in the Stockholm Convention were determined using isotope-dilution gas chromatography-high resolution mass spectrometry. This is the first study of hexachlorobenzene (HCB) in the Liaohe River basin sediments. The total OCP concentrations were 0.39-68.06 ng g(-1) dry weight. The total α-, ß-, γ-, and δ-hexachlorocyclohexane (HCH), the total dichlorodiphenyltrichloroethane (DDT - p,p'-dichlorodiphenyldichloroethane (DDD), p,p'-dichlorodiphenyldichloroethylene (DDE), o,p(')-DDT, and p,p'-DDT), and the HCB concentrations in the sediment samples were 0.1-28.48 ng g(-1) (mean 4.01 ng g(-1)), 0.08-6.52 ng g(-1) (mean 3.07 ng g(-1)), and 0.18-24.8 ng g(-1) (mean 4.38 ng g(-1)), respectively. The HCB concentrations were higher than the concentrations of the other OCPs, and the HCHs and HCB together were the dominant OCPs. ß-HCH was the most abundant HCH isomer. The concentrations of DDTs and other OCPs were relatively low, and the (DDE+DDD)/DDT ratios (>0.5) and DDD/DDE ratios (<1) indicated that no recent DDT inputs had occurred in the Liaohe River system. The main sources of HCHs were probably the historical production and agricultural use of HCH in the study area. The DDT and HCH concentrations were generally below or similar to the concentrations that have been found in other parts of the world. An ecotoxicological evaluation indicated that HCHs in surface sediments pose slight risks to human and ecological health in the Liaohe River basin.

Cannabinoid receptor agonist WIN55,212-2 and fatty acid amide hydrolase inhibitor URB597 suppress chronic cerebral hypoperfusion-induced neuronal apoptosis by inhibiting c-Jun N-terminal kinase signaling.

The endocannabinoid system (ECS) has therapeutic potential for treating chronic cerebral hypoperfusion (CCH)-induced cerebral diseases. This study investigated the protective effects of two ECS compounds, cannabinoid receptor agonist WIN55,212-2 (WIN) and fatty acid amide hydrolase inhibitor URB597 (URB) on CCH-induced neuronal apoptosis in vivo. CCH was induced in male Sprague-Dawley rats by bilateral common carotid artery occlusion (BCCAo); the rats were then treated with WIN or URB for 12weeks and their spatial learning and memory abilities were assessed using the Morris water maze. Changes in neuronal number were examined by labeling neurons with an antibody against the neuronal nuclei antigen, and apoptosis of cortical and hippocampal CA1 neurons was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling. The expression of B cell lymphoma (Bcl)-2, Bcl-2-associated X protein (Bax), and activated caspase-3 as well as mitogen-activated protein kinases including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38, phosphorylated (p-)ERK, p-JNK, and p-P38 was examined by Western blotting. Rats treated with WIN or URB showed better learning and memory performance than controls. The neuroprotective effects of URB were greater than those of WIN, and co-administration of WIN and URB had a synergistic effect. In addition, WIN and URB blocked JNK phosphorylation as well as the decrease in Bcl-2/Bax ratio and caspase-3 activation induced by CCH, implying that these agents modulate neuronal survival. Moreover, the selective JNK inhibitor SP600125 improved mitochondrial membrane dysfunction and blocked neuronal apoptosis induced by JNK-dependent Bcl-2 signaling. WIN and URB enhanced the effects of SP600125, implying that they may exert anti-apoptotic effects in part by inhibiting a non-nuclear JNK pathway. These findings indicate that WIN and URB promote neuronal survival and may potentially be used to protect neurons against chronic ischemic insults.

Design and synthesis of 3-isoxazolidone derivatives as new Chlamydia trachomatis inhibitors.

Chlamydia trachomatis (Ct) is a bacterial human pathogen responsible for the development of trachoma, the worldwide infection leading to blindness, and is also a major cause of sexually transmitted diseases. As iron is an essential metabolite for this bacterium, iron depletion presents a promising strategy to limit Ct proliferation. The aim of this study is to synthesize 3-isoxazolidone derivatives bearing known chelating moieties in an attempt to develop new bactericidal anti-Chlamydiaceae molecules. We have investigated the paths by which these new compounds affect Ct serovar L2 development in HeLa cells, in the presence or absence of exogenously added iron. The iron-chelating properties of these molecules were also determined. Our data reveal important bactericidal effects which are distinguishable from those due to iron chelation.

Community level offset of rain use- and transpiration efficiency for a heavily grazed ecosystem in inner Mongolia grassland.

Water use efficiency (WUE) is a key indicator to assess ecosystem adaptation to water stress. Rain use efficiency (RUE) is usually used as a proxy for WUE due to lack of transpiration data. Furthermore, RUE based on aboveground primary productivity (RUEANPP) is used to evaluate whole plant water use because root production data is often missing as well. However, it is controversial as to whether RUE is a reliable parameter to elucidate transpiration efficiency (TE), and whether RUEANPP is a suitable proxy for RUE of the whole plant basis. The experiment was conducted at three differently managed sites in the Inner Mongolia steppe: a site fenced since 1979 (UG79), a winter grazing site (WG) and a heavily grazed site (HG). Site HG had consistent lowest RUEANPP and RUE based on total net primary productivity (RUENPP). RUEANPP is a relatively good proxy at sites UG79 and WG, but less reliable for site HG. Similarly, RUEANPP is good predictor of transpiration efficiency based on aboveground net primary productivity (TEANPP) at sites UG79 and WG but not for site HG. However, if total net primary productivity is considered, RUENPP is good predictor of transpiration efficiency based on total net primary productivity (TENPP) for all sites. Although our measurements indicate decreased plant transpiration and consequentially decreasing RUE under heavy grazing, productivity was relatively compensated for with a higher TE. This offset between RUE and TE was even enhanced under water limited conditions and more evident when belowground net primary productivity (BNNP) was included. These findings suggest that BNPP should be considered when studies fucus on WUE of more intensively used grasslands. The consideration of the whole plant perspective and "real" WUE would partially revise our picture of system performance and therefore might affect the discussion on the C-sequestration and resilience potential of ecosystems.

Assessment of cognitive function in adult patients with hemorrhagic moyamoya disease who received no surgical revascularization.

BACKGROUND AND PURPOSE: Due to controversial surgical treatment for hemorrhagic moyamoya disease (MMD), a large proportion of these patients chose conservative treatment. The aim of this study was to assess cognitive function in adult patients with hemorrhagic MMD who received no surgical revascularization. METHODS: Twenty-six adult hemorrhagic MMD patients with only intraventricular hemorrhage (IVH) confirmed by positive computed tomography and magnetic resonance imaging scan, 20 patients with spontaneous IVH whose digital subtraction angiography results were negative, and 30 healthy controls were identified and matched for age, gender, education background and living area. Cognitive function was evaluated by Montreal Cognitive Assessment (MoCA). The non-parametric test was used for comparisons among the three groups. RESULTS: No patient was confirmed cognitive dysfunction at the initial screening. Twenty-four (92%) cases presented mild cognitive impairment (MCI) after 1 year. All the cases demonstrated MCI after 2 years. The difference between cases and healthy controls was statistically significant at the second screening (P = 0.000) and the third screening (P = 0.000), as was that between cases and patients with spontaneous IVH at the second screening (P = 0.000) and the third screening (P = 0.000). In addition, there were significant decreases in all MoCA subscores (P = 0.000) with special regards to delayed recall, visual space and executive function in cases compared with the other two groups. Moreover, significant differences were found in the subgroups of smoking (P = 0.021) and Suzuki angiographic classification of MMD (P = 0.030). CONCLUSIONS: Cognitive impairment is a long-term complication for adult hemorrhagic MMD patients who underwent conservative treatment.

Characterization and distribution of PAHs in surface sediments of Daliao River, China.

The concentrations of 16 priority pollutant PAHs in the samples of Daliao River were analyzed by a method based on Soxhlet extraction, solid-phase extraction clean-up and high performance liquid chromatography-programmable fluorescence/ultra violet detection. The total concentrations of PAHs in Daliao River ranged from 267.9 ng/g to 9,212 ng/g. The highest concentration of PAHs was 9,212 ng/g at the Station 24 (Anshan Railroad Bridge), and the lowest 267.9 ng/g at the Station 19 (Estuary). Compared with the total concentrations of PAHs of rivers in other parts of the world, the degree of contamination of Daliao River by PAHs was moderate to high. Specific compounds or groups of PAHs have been used as molecular markers to differentiate petrogenic and pyrogenic origin. The results showed that the main sources of PAHs in Daliao River were pyrolytic inputs such as combustion of coal, wood and petrogenic chemicals. The data was also compared by mean of two guideline values, an effects range-low (ER-L) and effects range-medium (ER-M), to assess the potential biological effects of the sediment adsorbed PAHs. It showed that the PAHs in some sites of Daliao River would exert adverse biological effects.

Response of hairy cells to IFN-alpha involves induction of apoptosis through autocrine TNF-alpha and protection by adhesion.

Although hairy cell leukemia is uniquely sensitive to interferon-alpha (IFN-alpha), the biologic basis for this phenomenon remains unclear. Here we examine the effects of IFN-alpha on cultured hairy cells (HCs), taking into account the possible modifying influence of cell adhesion. We make the novel observation that therapeutic concentrations of IFN-alpha kill nonadherent HCs by inducing apoptosis. In keeping with the persistence of HCs in tissues during therapy, such killing was inhibited by integrin-mediated adhesion to vitronectin or fibronectin. Exposure of HCs to IFN-alpha resulted in a marked increase in tumor necrosis factor-alpha (TNF-alpha) secretion. Furthermore, blocking antibodies to TNF-RI or TNF-RII protected HCs from IFN-alpha-induced apoptosis, demonstrating that such killing was mediated by TNF-alpha. In the absence of IFN-alpha, exogenous TNF-alpha did not induce HC apoptosis, showing that IFN-alpha sensitized HCs to the proapoptotic effect of autocrine TNF-alpha. This sensitization to TNF-alpha-induced killing was attributable to suppression of IAP (inhibitors of apoptosis) production known to be regulated by the cytoprotective nuclear factor-kappaB-dependent arm of TNF-alpha signaling. Moreover, engagement of the receptors for fibronectin or vitronectin prevented this IFN-alpha-induced down-regulation of IAPs. Understanding of the signals involved in the combined effects of IFN-alpha and TNF-alpha and abrogation of those induced by integrin engagement offers the possibility of sensitizing other malignant cells to IFN-alpha-induced killing and thereby extending the therapeutic use of this cytokine.

Image-guided radiosurgery for the spine and pancreas.

A robotic image-guided radiosurgical system has been modified to treat extra-cranial sites using implanted fiducials and skeletal landmarks to locate the treatment targets. The system has been used to treat an artero-venous malformation in the cervical spine, a recurrent schwannoma of the thoracic spine, a metastatic adenocarcinoma of the lumbar spine, and three pancreatic cancers. During each treatment, the image guidance system monitored the position of the target site and relayed the target coordinates to the beam-pointing system at discrete intervals. The pointing system then dynamically aligned the therapy beam with the lesion, automatically compensating for shifts in target position. Breathing-related motion of the pancreas lesions was managed by coordinating beam gating with breath-holding by the patient. The system maintained alignment with the spine lesions to within +/- 0.2 mm on average, and to within +/- 1 mm for the pancreatic tumors. This experience has demonstrated the feasibility of using image-guided robotic radiosurgery outside the cranium.