Molecular Alterations of Circulating Cell-Free DNA in the Pathological Progression of Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most malignant cancers. Early diagnosis of HCC is important to reduce the mortality rate. The aim of this study is to explore the plasma cell-free DNA (cfDNA) mutation profile in the pathological progression of HCC and to investigate the significance of plasma cfDNA mutations in the early diagnosis of HCC. METHODS: Thirty-seven patients with chronic hepatitis B (CHB), eight with liver cirrhosis (LC), and eleven with HCC were enrolled in this cohort. Plasma cfDNA and white blood cell DNA were isolated, and plasma cfDNA mutation profiles were detected using a targeted gene panel. RESULTS: The sequencing results of plasma cfDNA showed that HCC-related gene mutations were present in patients with CHB and LC. The mutation burden of HCC-related genes increased from CHB and LC to HCC. In patients with HCC, the average mutation burden of NRAS (10.1%), TP53 (7.4%), PTEN (4.2%), and APOB (2.6%) was the highest. The average mutation burden of PTEN, APOB, FRAS1, KDM6A, DDR2, TTK, NRAS, TP53, PTPRB, MPL, FCRL1, HN1, and SFN gradually increased from CHB and LC to HCC. The mutation burden of 18 HCC-related genes had an area under the receiver operating characteristics of 0.92 for the diagnosis of HCC. CONCLUSIONS: The mutation burden of HCC-related genes increased from CHB and LC to HCC. An optimal combination of cfDNA mutations in the gene panel for diagnosing HCC in patients with CHB and LC was selected. Our study indicates that somatic mutations in plasma cfDNA may serve as potential biomarkers for early HCC diagnosis.

A Machine Learning Approach Yields a Multiparameter Prognostic Marker in Liver Cancer.

A number of staging systems have been developed to predict clinical outcomes in hepatocellular carcinoma (HCC). However, no general consensus has been reached regarding the optimal model. New approaches such as machine learning (ML) strategies are powerful tools for incorporating risk factors from multiple platforms. We retrospectively reviewed the baseline information, including clinicopathologic characteristics, laboratory parameters, and peripheral immune features reflecting T-cell function, from three HCC cohorts. A gradient-boosting survival (GBS) classifier was trained with prognosis-related variables in the training dataset and validated in two independent cohorts. We constructed a 20-feature GBS model classifier incorporating one clinical feature, 14 laboratory parameters, and five T-cell function parameters obtained from peripheral blood mononuclear cells. The GBS model-derived risk scores demonstrated high concordance indexes (C-indexes): 0.844, 0.827, and 0.806 in the training set and validation sets 1 and 2, respectively. The GBS classifier could separate patients into high-, medium- and low-risk subgroups with respect to death in all datasets (P < 0.05 for all comparisons). A higher risk score was positively correlated with a higher clinical stage and the presence of portal vein tumor thrombus (PVTT). Subgroup analyses with respect to Child-Pugh class, Barcelona Clinic Liver Cancer stage, and PVTT status supported the prognostic relevance of the GBS-derived risk algorithm independent of the conventional tumor staging system. In summary, a multiparameter ML algorithm incorporating clinical characteristics, laboratory parameters, and peripheral immune signatures offers a different approach to identify patients with the greatest risk of HCC-related death.

Anticancer activity, topoisomerase I inhibition, DNA 'light switch' behavior and molecular docking of two ruthenium complexes containing phenazine ring.

Ruthenium(II) complexes containing phenazine ring have attracted attention to design as 'molecular light switches'. In this study, we synthesized two ruthenium complexes containing phenazine ring and studied their abilities to function as DNA intercalators, DNA 'light switches', DNA topo I inhibitors, DNA photocleavers and potential antitumor reagents. [Ru(bpy)2(mbipz)](PF6)2 (1) (bpy = 2,2'-bipyridine, mbipz = 2-(4'-methyl-bipyridine-4-yl)-1H-imidazo[4,5-b]phenazine) exhibited off-on type DNA 'light swtich' behavior. DNA binding modes of the two complexes were determined as intercalation by using UV-vis spectra, emission spectra, viscosity and molecular docking experiments. DNA photocleavage experimental results showed that the two ruthenium complexes effectively cleave plasmid DNA by producing singlet oxygen. Furthermore, they displayed good topo I inhibition activities. We further found that the two complexes displayed good antitumor activities against Eca-109 cells and A549 cells. The results demonstrated that introduction of phenazine ring will be helpful to design DNA 'light switch' based on ruthenium complex.Communicated by Ramaswamy H. Sarma.

Nitro-Substituted Ruthenium(II) Complex: A New Strategy for a G-Quadruplex DNA Fluorescent Probe.

A new strategy for a G-quadruplex fluorescent probe based on a nitro-substituted ruthenium complex is described. G-quadruplex DNA can be distinguished from double- or single-strand DNA by the naked eye. This ability originates from variation of the degree of protection of the nitro group on the complex from water by G-quadruplex and other structure DNAs.

Early-phase peritoneal drainage and lavage in a rat model of severe acute pancreatitis.

PURPOSE: To evaluate the effects of early-phase drainage on the survival rates and pancreatic pathological changes associated with severe acute pancreatitis (SAP) in a rat model. METHODS: Sprague-Dawley rats were divided into the following groups: SAP model (control), early drainage and delayed drainage. The 24-h survival rates were compared among the groups. In addition, the serum and ascites concentrations of interleukin (IL)-1ß, IL-6, IL-8, IL-10 and tumor necrosis factor (TNF)-α were measured, and pancreatic pathological changes were observed. RESULTS: The survival rate significantly improved in the early drainage group. Compared with that observed in the control group, the serum TNF-α and IL-8 concentrations in the early drainage group decreased, while the serum IL-10 levels increased, and the ascites concentrations of IL-1ß, IL-6, IL-8 and TNF-α decreased, while that of IL-10 increased significantly. In the delayed drainage group, only the ascites concentrations of TNF-α decreased. Meanwhile, the pancreatic pathological changes at 3, 6 and 24 h worsened in the early drainage group; however, the pancreatic lesions in the early drainage group were less mild than those seen in the control group. CONCLUSIONS: Rebalancing the cytokine levels in ascites after early drainage may be a key factor for enhancing the survival rate in rats.

DNA Interaction, Photocleavage and Topoisomerase I Inhibition by Ru(II) Complex with a New Ligand Possessing Phenazine Unit.

A new ruthenium complex with a dppz-like ligand pyidppz, [Ru(bpy)2(pyidppz)](2+) (pyidppz = 2-(pyridine-2-yl)imidazo-[4,5-b]dipyrido-[3,2-a:2',3'-c]phenazine) has been synthesized and characterized by ES-MS, elemental analysis, (1)H NMR. Intercalative mode of the complex bound to calf thymus DNA has been supported by different spectroscopic methods and viscosity measurements. The introduction of phenazine unit may be one of the main reasons for the weak emission of Ru(II) complex in aqueous solution. Under irradiation, this complex can efficiently cleave DNA. And the photocleavage reaction of the complex is found to be inhibited in the presence of singlet oxygen scavenger. Topoisomerase inhibition and DNA strand passage assay demonstrated that [Ru(bpy)2(pyidppz)](2+) and its parent complex [Ru(bpy)2(pyip)](2+) (pyip = 2-(pyridine-2-yl)imidazo[4,5-f][1,10]phenanthroline) can act as efficient catalytic inhibitor of DNA topoisomerase I.

Background eliminated signal-on electrochemical aptasensing platform for highly sensitive detection of protein.

Using platelet-derived growth factor B chain dimer (PDGF-BB) as the model target, a background current eliminated electrochemical aptameric sensing platform for highly sensitive and signal-on detection of protein is proposed in this paper. Successful fabrication of the biosensor depends on ingenious design of aptamer probe, which contains the aptamer sequence for PDGF-BB and the recognition sequence for EcoRI endonuclease. In the absence of PDGF-BB, the ferrocene labeled aptamer probe folds into a hairpin structure and forms a recognition site for EcoRI. By treatment with endonuclease, the specific and cleavable double-stranded region is cut off and redox-active ferrocene molecule is removed from the electrode surface, and almost no peak current is observed. When binding with target protein, the designed aptamer probe changes its conformation and dissociates the recognition double strand. The integrated aptamer probe is maintained when exposing to EcoRI endonuclease, resulting in obvious peak current. Therefore, a signal-on and sensitive sensing strategy for PDGF-BB detection is fabricated with eliminated background current. Under the optimized experimental conditions, a wide linear response range of 4 orders of magnitude from 20pgmL(-1) to 200ngmL(-1) is achieved with a detection limit of 10pgmL(-1). Moreover, the present aptameric platform is universal for the analysis of a broad range of target molecules of interest by changing and designing the sequence of aptamer probe.

DNA interaction and photocleavage properties of Ru (II) complexes [Ru(bpy)2(pibi)]²âº and [Ru(phen)2(pibi)]²âº.

A new asymmetry ligand pibi (pibi = 2-(pyridine-2-yl)-1-H-imidazo[4,5-f]benzo[d]imidazolone) and its ruthenium complexes with [Ru(L)2(pibi)](2+) (L = bpy (2, 2'-bipyridine), phen (1, 10-phenanthroline)), have been synthesized and characterized. The binding of two complexes with calf thymus DNA has been investigated by spectroscopic and viscosity measurement. The results indicate that both complexes can bind to CT-DNA through intercalative mode. Under irradiation at 365 nm, both complexes can partly promote the photocleavage of plasmid pBR322DNA. The low singlet oxygen generation abilities of the two complexes may be the factor for the low DNA photocleavage abilities.

A novel model for orthotopic liver transplantation in rats using hepatic rearterialization and biliary extradrainage system.

BACKGROUND: Although the rat orthotopic liver transplantation (OLT) model has existed for many years, only a few models can be applied for dynamic bile collection. The aim of this study was to introduce a dependent rat OLT model with hepatic rearterialization and an expediently dynamic bile collection system. METHODS: Forty-five male Sprague-Dawley rats were divided into the following three groups (n = 15 each): group A, OLT without hepatic rearterialization; group B, OLT with hepatic rearterialization; group C, OLT with hepatic rearterialization and a biliary extradrainage system. In groups B and C, a modified sleeve anastomosis between the donor common hepatic artery and the recipient proper hepatic artery was performed to restore the hepatic artery blood flow. In group C, after hepatic rearterialization, biliary extradrainage and jejunum stoma were performed to reestablish the bile flow, and a waistcoat-like external fixator was introduced to protect this system. RESULTS: The surgical success rates in groups A, B, and C were 100% (15/15), 93% (14/15), and 93% (14/15), respectively. In groups B and C, the hepatic artery patency rates were 93% and 86% on postoperative day 3 and postoperative day 21, respectively. Also, the liver function and bile duct integrity were preserved better than that in group A. In group C, the biliary extradrainage system was well preserved and bile collection was easily performed. CONCLUSIONS: The rat OLT model with hepatic rearterialization and a convenient biliary extradrainage system was satisfactory in maintaining the survival rate, hepatic artery patency rate, and recovery of graft function, so it can be applied in various studies after transplantation.

DNA-binding, photocleavage studies of ruthenium(II) complexes with 2-(2-quinolinyl) imidazo[4,5-f][1,10]phenanthroline.

Two new ruthenium complexes with [Ru(L)(2)(qip)](2+) (L=bpy (2,2'- bipyridine), phen (1,10-phenanthroline); qip=2-(2-quinolinyl)imidazo[4,5-f][1,10]phenanthroline), have been synthesized and characterized by elemental analysis, ES-MS, (1)H NMR. The binding properties of two complexes towards CT-DNA were investigated by various optical methods and viscosity measurements. The experiment results suggested that both Ru(II) complexes can intercalate into DNA base pairs. Strong quenching in emission intensity of two Ru(II) complexes were observed upon addition of Ag(+) in the absence and presence of CT-DNA. Furthermore, the two complexes can promote cleavage of pBR322 DNA under irradiation at 365 nm, and complex 2 exhibits a stronger DNA-photocleavage efficiency than complex 1. The mechanism of DNA cleavage suggests that singlet oxygen ((1)O(2)) is likely to be the cleaving agent.

DNA-binding and photocleavage studies of ruthenium(II) complexes containing asymmetric intercalative ligand.

A novel asymmetric ligand 2-(pyridine-2-yl)-1-H-imidazo[4,5-i]dibenzo[2,3-a:2',3'-c]phenazine (pidbp) and its ruthenium complexes [Ru(L)(2)(pidbp)](2+) (L=bpy (2, 2'- bipyridine), phen (1, 10 - phenanthroline)), have been synthesized and characterized by elemental analysis, ES-MS, (1)H NMR. Various methods support the conclusion that both Ru(II) complexes can intercalate into DNA base pairs. Complex [Ru(bpy)(2)(pidbp)](2+)4 exhibits its DNA "molecular light switch" properties. Furthermore, the two complexes are efficient DNA-photocleavers under irradiation at 365 nm, and complex 5 exhibits a stronger DNA-photocleavage efficiency than complex 4. The mechanism of DNA cleavage is an oxidative process by generating singlet oxygen.

The fabrication of a piezoelectric immunosensor based on DNA-antibody conjugate layer.

In this article, we report a method of antibody immobilization carried out by hybridizing DNA-antibody conjugates on a mixed self-assembled monolayer composed of DNA thiols and mercaptopropionic acid via sequence-specific hybridization. The proposed method was applied to fabricate an immunosensor for detecting human immunoglobulin G (IgG). Under the optimized experimental conditions, a wide linear range from 50.0 to 500 µg/ml was reached with a detection limit of 30.13 µg/ml. The developed immunosensor possesses advantages such as simple fabrication, wide linear range, easy regeneration, and excellent reproducibility.

Overexpression of Forkhead box M1 protein associates with aggressive tumor features and poor prognosis of hepatocellular carcinoma.

The aim of this study was to detect the expression of the Forkhead box M1 (FOXM1) protein in human hepatocellular carcinoma (HCC) and to associate FOXM1 expression with clinicopathological features of the patients, and predict the prognosis of patients with FOXM1 expression. Surgical tissue specimens from 151 HCC patients were subjected to a tissue microarray construction and immunohistochemistry analysis of FOXM1 and the proliferation marker proliferating cell nuclear antigen (PCNA). The data showed that the FOXM1 protein was expressed in 59.3% of the HCC tissues, which was significantly higher compared to that of the surrounding non-tumorous tissues (23.8%; P<0.001). Moreover, FOXM1 expression was positively correlated with the labeling index of PCNA (P<0.001) in HCC and with aggressive tumor phenotypes, such as larger tumor size, multiple tumors, bilobar involvement, poor tumor cell differentiation, advanced stage and macrovascular invasion (P<0.05). In addition, HCC patients with FOXM1-positive tumors had a poorer recurrence-free and overall survival after hepatectomy than those with FOXM1-negative tumors. Multivariate Cox regression analysis demonstrated that FOXM1 expression was an independent predictor of unfavorable outcome (P<0.05). The data from the current study suggest that FOXM1 may play an important role in HCC progression and could be further evaluated as a prognostic biomarker and potential therapeutic target.

Improvement of antibody immobilization using hyperbranched polymer and protein A.

For the construction of a well-defined antibody surface, protein A was used as a binding material to immobilize antibodies onto gold-derivatized transducers. The traditional method tends to assemble protein A directly onto the gold-derivatized transducers. In this paper, we tried to indirectly bind protein A onto sensors through hyperbranched polymer (HBP) which was synthesized from p-phenylenediamine and trimesic acid. The three-dimensional structure of HBP and the characteristics including orientation control and biocompatibility of protein A led to highly efficient immunoreactions and enhanced detection system performance. With this strategy, cysteamine monolayer was first assembled onto Au electrodes associated with the piezoelectric quartz crystal; secondly, the cysteamine-modified gold electrode was further modified by the activated HBP; thirdly, protein A was immobilized onto the HBP film; and finally, antibodies were immobilized onto the surface of protein A film for detecting the corresponding antigen. The quartz crystal microbalance immunosensor thus fabricated was applied to detect hepatitis B surface antigen in solutions that ranged from 0.71 to 300 µg mL(-1). The detection limit was estimated to be 0.53 µg mL(-1). The immunosensor holds good selectivity, sensitivity, and repeatability.

N,N'-Bis(pyridin-3-yl)terephthalamide-terephthalic acid (1/1).

In the title compound, C(18)H(14)N(4)O(2)·C(8)H(6)O(4), both types of mol-ecule lie on inversion centers. In the N,N'-bis-(pyridin-3-yl)terephthalamide mol-ecule, the pyridine ring forms a dihedral angle of 11.33 (9)° with the central benzene ring. In the crystal, N-H⋯O and O-H⋯N hydrogen bonds connect the components into a three-dimensional network.

Synthesis, DNA-binding and photocleavage of "light switch" complexes [Ru(bpy)2(pyip)]2+ and [Ru(phen)2(pyip)]2+.

Two novel Ru(II) complexes [Ru(bpy)(2)(pyip)](2+)1 and [Ru(phen)(2)(pyip)](2+)2 (bpy=2,2'-bipyridine; phen=1,10-phenanthroline; pyip=2-(pyridine-2-yl)imidazo-[4,5-f][1,10]-phenanthroline), have been synthesized and characterized by elemental analysis, ES-MS, (1)H NMR, UV-Vis. The DNA-binding behaviors of both complexes were studied by spectroscopic methods and viscosity measurements. The results indicate that the two complexes can bind to CT-DNA in an intercalative mode, and also show that these two Ru(II) complexes can promote the photocleavage of pBR322 DNA. In addition, In the presence of Co(2+), the emission of DNA-[Ru(L)(2)pyip](2+) can be quenched, which exhibited the DNA "light switch" properties.

A novel piezoelectric quartz crystal immnuosensor based on hyperbranched polymer films for the detection of alpha-Fetoprotein.

This paper reports a novel piezoelectric quartz crystal immnuosensor based on hyperbranched polymer films for the detection of alpha-Fetoprotein. In this strategy, the sensing interfaces consist of a primary cystamine monolayer assembled onto Au electrodes associated with the piezoelectric quartz crystal. The monolayer is further modified with a new hyperbranched polymer which was synthesized through direct polycondensation of monomer 5-[3-(4-aminophenyl) propionylamino] isophthalic acid. The detection performances of resulting immunosensor were investigated by use of the antibody-antigen model system of alpha-Fetoprotein (AFP), an important indicator in the diagnosis of clinical cancers. The analytical technique is characterised through the investigation of different methods of assembling the monolayers used as supports, as well as by comparing two different types of supports. It was found that the developed sensing interface could perform more effectively in antibody-antigen binding and consequently increased the sensitivity of the whole piezoelectric immunosensor. Moreover, the method should also be useful for the construction of other kind of immunosensors.

Electrochemical properties of Nile Blue covalently immobilized on self-assembled thiol-monolayer modified gold electrodes.

A monolayer of Nile Blue (NB) has been covalently immobilized on the self-assembled thiol-monolayer modified gold electrode. Cyclic voltammograms indicated a stable and reverse redox process of NB bonded on the electrode surface. The mechanisms of redox process coupling with proton transfer were proposed. The NB-modified electrode showed excellent electrocatalytic activity toward Nicotinamide adenine dinucleotide (NADH) oxidation and horseradish peroxidase (HRP) reduction. A hydrogen peroxide biosensor based on NB as a mediator has been demonstrated.