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1.  Virus-related liver cirrhosis: Molecular basis and therapeutic options 
Chronic infections with hepatitis B virus (HBV) and/or hepatitis C virus (HCV) are the major causes of cirrhosis globally. It takes 10-20 years to progress from viral hepatitis to cirrhosis. Intermediately active hepatic inflammation caused by the infections contributes to the inflammation-necrosis-regeneration process, ultimately cirrhosis. CD8+ T cells and NK cells cause liver damage via targeting the infected hepatocytes directly and releasing pro-inflammatory cytokine/chemokines. Hepatic stellate cells play an active role in fibrogenesis via secreting fibrosis-related factors. Under the inflammatory microenvironment, the viruses experience mutation-selection-adaptation to evade immune clearance. However, immune selection of some HBV mutations in the evolution towards cirrhosis seems different from that towards hepatocellular carcinoma. As viral replication is an important driving force of cirrhosis pathogenesis, antiviral treatment with nucleos(t)ide analogs is generally effective in halting the progression of cirrhosis, improving liver function and reducing the morbidity of decompensated cirrhosis caused by chronic HBV infection. Interferon-α plus ribavirin and/or the direct acting antivirals such as Vaniprevir are effective for compensated cirrhosis caused by chronic HCV infection. The standard of care for the treatment of HCV-related cirrhosis with interferon-α plus ribavirin should consider the genotypes of IL-28B. Understanding the mechanism of fibrogenesis and hepatocyte regeneration will facilitate the development of novel therapies for decompensated cirrhosis.
doi:10.3748/wjg.v20.i21.6457
PMCID: PMC4047331  PMID: 24914367
Liver cirrhosis; Hepatitis B virus; Hepatitis C virus; Evolution; Immune cells; Signaling pathway; Hepatic stellate cells; Antiviral therapy
2.  Solitary schwannoma of the gallbladder: A case report and literature review 
Schwannomas occurring in the gallbladder are extremely rare. Preoperative diagnosis of gallbladder schwannomas appears to be very difficult because they are normally asymptomatic and are often found incidentally. Until now, only five cases have been reported in the literature. To our knowledge, the contrast-enhanced ultrasound (CEUS) features of gallbladder schwannomas have not been reported before in other studies. We treated a 55-year-old male patient with gallbladder schwannoma in China. He had no symptoms, and the lesion was incidentally found by conventional ultrasound (US) when performing a health examination. The patient had normal liver function; moreover, serum carcinoembryonic antigen and alpha-fetoprotein were within the normal ranges. The lesion showed no blood flow signals on color Doppler US, and the wall beneath the lesion was intact on CEUS. The lesion was believed to be a benign entity; in addition, gallbladder adenomyomatosis was suspected. A laparoscopic cholecystectomy was performed to remove the mass. Pathological examination revealed that the tumor was mainly composed of spindle-shaped cells; neither atypical cells nor signs of malignancy were found. Immunohistochemical staining showed a strong positive S-100 protein reaction. Vimentin and CD56 staining were also positive, whereas CD34 and CD117 were negative. Finally, the lesion was diagnosed as schwannoma. Herein, we report the case; the associated literature is also reviewed.
doi:10.3748/wjg.v20.i21.6685
PMCID: PMC4047360  PMID: 24914396
Schwannoma; Gallbladder; Ultrasound; Contrast-enhanced ultrasound
3.  Follicle-stimulating hormone enhances the proliferation of ovarian cancer cells by activating transient receptor potential channel C3 
Endocrine-related cancer  2013;20(3):415-429.
Recent studies have suggested that follicle-stimulating hormone (FSH) plays an important role in ovarian epithelial carcinogenesis. We demonstrated that FSH stimulates the proliferation and invasion of ovarian cancer cells, inhibits apoptosis, and facilitates neovascularisation. Our previous work has shown that transient receptor potential channel C3 (TRPC3) contributes to the progression of human ovarian cancer. In this study, we further investigated the interaction between FSH and TRPC3. We found that FSH stimulation enhanced the expression of TRPC3 at both the mRNA and protein levels. SiRNA-mediated silencing of TRPC3 expression inhibited the ability of FSH to stimulate proliferation and blocked apoptosis in ovarian cancer cell lines. FSH stimulation was associated with the upregulation of TRPC3, while also facilitating the influx of Ca2+ after treatment with a TRPC-specific agonist. Knockdown of TRPC3 abrogated FSH-stimulated Akt/PKB phosphorylation, leading to decreased expression of downstream effectors including survivin, HIF1α and VEGF. Ovarian cancer specimens were analysed for TRPC3 expression; higher TRPC3 expression levels correlated with early relapse and worse prognosis. Association with poor disease-free survival and overall survival remained after adjusting for clinical stage and grade. In conclusion, TRPC3 plays a significant role in the stimulating activity of FSH and could be a potential therapeutic target for the treatment of ovarian cancer, particularly in postmenopausal women with elevated FSH levels.
doi:10.1530/ERC-12-0005
PMCID: PMC3669658  PMID: 23580589
ovarian epithelial cancer; follicle-stimulating hormone (FSH); transient receptor potential channel C3 (TRPC3); cell proliferation; prognosis
4.  PKA Type IIα Holoenzyme Reveals a Combinatorial Strategy for Isoform Diversity 
Science (New York, N.Y.)  2007;318(5848):274-279.
The catalytic (C) subunit of cyclic adenosine monophosphate (cAMP)–dependent protein kinase (PKA) is inhibited by two classes of regulatory subunits, RI and RII. The RII subunits are substrates as well as inhibitors and do not require adenosine triphosphate (ATP) to form holoenzyme, which distinguishes them from RI subunits. To understand the molecular basis for isoform diversity, we solved the crystal structure of an RIIα holoenzyme and compared it to the RIα holoenzyme. Unphosphorylated RIIα (90–400), a deletion mutant, undergoes major conformational changes as both of the cAMP-binding domains wrap around the C subunit’s large lobe. The hallmark of this conformational reorganization is the helix switch in domain A. The C subunit is in an open conformation, and its carboxyl-terminal tail is disordered. This structure demonstrates the conserved and isoform-specific features of RI and RII and the importance of ATP, and also provides a new paradigm for designing isoform-specific activators or antagonists for PKA.
doi:10.1126/science.1146447
PMCID: PMC4036697  PMID: 17932298
5.  In situ precipitation: a novel approach for preparation of iron-oxide magnetoliposomes 
Background
Conventional methods of preparing magnetoliposomes are complicated and inefficient. A novel approach for magnetoliposomes preparation was investigated in the study reported here.
Methods
FeCl3/FeCl2 solutions were hydrated with lipid films to obtain liposome-encapsulated iron ions by ultrasonic dispersion. Non-encapsulated iron ions were removed by dialysis. NH3 · H2O was added to the system to adjust the pH to a critical value. Four different systems were prepared. Each was incubated at a different temperature for a different length of time to facilitate the permeation of NH3 · H2O into the inner phase of the liposomes and the in situ formation of magnetic iron-oxide cores in the liposomes. Single-factor analysis and orthogonal-design experiments were applied to determinate the effects of alkalization pH, temperature, duration, and initial Fe concentration on encapsulation efficiency and drug loading.
Results
The magnetoliposomes prepared by in situ precipitation had an average particle size of 168±14 nm, zeta potential of −26.2±1.9 mV and polydispersity index of 0.23±0.06. The iron-oxide cores were confirmed as Fe3O4 by X-ray diffraction and demonstrated a superparamagnetic response. Encapsulation efficiency ranged from 3% to 22%, while drug loading ranged from 0.2 to 1.58 mol Fe/mol lipid. The optimal conditions for in situ precipitation were found to be an alkalization pH of 12, temperature of 60°C, time of 60 minutes, and initial Fe concentration of 100 mM Fe3+ + 50 mM Fe2+.
Conclusion
In situ precipitation could be a simple and efficient approach for the preparation of iron-oxide magnetoliposomes.
doi:10.2147/IJN.S59859
PMCID: PMC4043714  PMID: 24920898
magnetoliposomes; in situ precipitation; iron oxide; alkalization; permeability
6.  Down-regulated γ-catenin expression is associated with tumor aggressiveness in esophageal cancer 
AIM: To evaluate the significance of γ-catenin in clinical pathology, cellular function and signaling mechanism in esophageal squamous cell carcinoma (ESCC).
METHODS: The mRNA expression of γ-catenin was detected by real-time quantitative reverse transcription-polymerase chain reaction in 95 tissue specimens and evaluated for association with the clinicopathologic characteristics and survival time of patients with ESCC. siRNAs against human γ-catenin were used to inhibit γ-catenin expression. Hanging drop aggregation assay and dispase-based dissociation assay were performed to detect the effect of γ-catenin on ESCC cell-cell adhesion. Transwell assay was performed to determine cell migration. Luciferase-based transcriptional reporter assay (TOPflash) was used to measure β-catenin-dependent transcription in cells with reduced γ-catenin expression. The expression and subcellular localizations of β-catenin and E-cadherin were examined using Western blot and immunofluorescence analysis.
RESULTS: γ-catenin mRNA expression was significantly associated with tumor histological grade (P = 0.017) in ESCC. Kaplan-Meier survival analysis showed that γ-catenin expression levels had an impact on the survival curve, with low γ-catenin indicating worse survival (P = 0.003). The multivariate Cox regression analysis demonstrated that γ-catenin was an independent prognostic factor for survival. Experimentally, silencing γ-catenin caused defects in cell-cell adhesion and a concomitant increase in cell migration in both KYSE150 and TE3 ESCC cells. Analysis of Wnt signaling revealed no activation event associated with γ-catenin expression. Total β-catenin and Triton X-100-insoluble β-catenin were significantly reduced in the γ-catenin-specific siRNA-transfected KYSE150 and TE3 cells, whereas Triton X-100-soluble β-catenin was not altered. Moreover, knocking down γ-catenin expression resulted in a significant decrease of E-cadherin and Triton X-100-insoluble desmocollin-2, along with reduced β-catenin and E-cadherin membrane localization in ESCC cells.
CONCLUSION: γ-catenin is a tumor suppressor in ESCC and may serve as a prognostic marker. Dysregulated expression of γ-catenin may play important roles in ESCC progression.
doi:10.3748/wjg.v20.i19.5839
PMCID: PMC4024793  PMID: 24914344
γ-catenin; Esophageal squamous cell carcinoma; Independent prognostic factor; Cell-cell adhesion; Cell migration
7.  Downregulation of microRNA-182-5p contributes to renal cell carcinoma proliferation via activating the AKT/FOXO3a signaling pathway 
Molecular Cancer  2014;13:109.
Background
Emerging evidence has suggested that dysregulation of miR-182-5p may contribute to tumor development and progression in several types of human cancers. However, its role in renal cell carcinoma (RCC) is still unknown.
Methods
Quantitative RT-PCR was used to quantify miR-182-5p expression in RCC clinical tissues. Bisulfite sequencing PCR was used for DNA methylation analysis. The CCK-8, colony formation, flow cytometry, and a xenograft model were performed. Immunohistochemistry was conducted using the peroxidase and DAB methods. A miR-182-5p target was determined by luciferase reporter assays, quantitative RT-PCR, and Western blotting.
Results
miR-182-5p is frequently down-regulated in human RCC tissues. Epigenetic modulation may be involved in the regulation of miR-182-5p expression. Enforced expression of miR-182-5p in RCC cells significantly inhibited the proliferation and tumorigenicity in vitro and in vivo. Additionally, overexpression of miR-182-5p induced G1-phase arrest via inhibition of AKT/FOXO3a signaling. Moreover, FLOT1 was confirmed as a target of miR-182-5p. Silencing FLOT1 by small interfering RNAs phenocopied the effects of miR-182-5p overexpression, whereas restoration of FLOT1 in miR-182-5p -overexpressed RCC cells partly reversed the suppressive effects of miR-182-5p.
Conclusions
These findings highlight an important role for miR-182-5p in the pathogenesis of RCC, and restoration of miR-182-5p could be considered as a potential therapeutic strategy for RCC therapy.
doi:10.1186/1476-4598-13-109
PMCID: PMC4040501  PMID: 24886554
Renal cell carcinoma; Proliferation; Microrna-182-5p; FLOT1
8.  Detection of Circulating Tumor DNA in Early- and Late-Stage Human Malignancies 
Science translational medicine  2014;6(224):224ra24.
The development of noninvasive methods to detect and monitor tumors continues to be a major challenge in oncology. We used digital polymerase chain reaction–based technologies to evaluate the ability of circulating tumor DNA (ctDNA) to detect tumors in 640 patients with various cancer types. We found that ctDNA was detectable in >75% of patients with advanced pancreatic, ovarian, colorectal, bladder, gastroesophageal, breast, melanoma, hepatocellular, and head and neck cancers, but in less than 50% of primary brain, renal, prostate, or thyroid cancers. In patients with localized tumors, ctDNA was detected in 73, 57, 48, and 50% of patients with colorectal cancer, gastroesophageal cancer, pancreatic cancer, and breast adenocarcinoma, respectively. ctDNA was often present in patients without detectable circulating tumor cells, suggesting that these two biomarkers are distinct entities. In a separate panel of 206 patients with metastatic colorectal cancers, we showed that the sensitivity of ctDNA for detection of clinically relevant KRAS gene mutations was 87.2% and its specificity was 99.2%. Finally, we assessed whether ctDNA could provide clues into the mechanisms underlying resistance to epidermal growth factor receptor blockade in 24 patients who objectively responded to therapy but subsequently relapsed. Twenty-three (96%) of these patients developed one or more mutations in genes involved in the mitogen-activated protein kinase pathway. Together, these data suggest that ctDNA is a broadly applicable, sensitive, and specific biomarker that can be used for a variety of clinical and research purposes in patients with multiple different types of cancer.
doi:10.1126/scitranslmed.3007094
PMCID: PMC4017867  PMID: 24553385
9.  BAG3 and HIF-1α Coexpression Detected by Immunohistochemistry Correlated with Prognosis in Hepatocellular Carcinoma after Liver Transplantation 
BioMed Research International  2014;2014:516518.
Objective. The objective is to determine the effects of BAG3 and HIF-1α expression on the prognosis of HCC patients after liver transplantation. Methods. Samples from 31 patients with HCC receiving liver transplantation were collected for this study. The immunohistochemistry was used to detect the expression of BAG3 and HIF-1α of HCC samples. Results. According to the immunohistochemistry results, BAG3 and HIF-1α staining were significantly associated with tumor TNM stage (P = 0.004, P = 0.012). A significant association between high BAG3/HIF-1α levels and a shorter overall survival was detected, so as the combined BAG3 and HIF-1α analysis. Conclusion. The results suggested that the expression level of BAG3 and HIF-1α is efficient prognostic parameters in patients with HCC after liver transplantation.
doi:10.1155/2014/516518
PMCID: PMC4034479  PMID: 24895585
10.  Clinical and computed tomography features of adult abdominopelvic desmoplastic small round cell tumor 
To investigate the clinical and computed tomography (CT) features of desmoplastic small round cell tumor (DSRCT), we retrospectively analyzed the clinical presentations, treatment and outcome, as well as CT manifestations of four cases of DSRCT confirmed by surgery and pathology. The CT manifestations of DSRCT were as follows: (1) multiple soft-tissue masses or diffuse peritoneal thickening in the abdomen and pelvis, with the dominant mass usually located in the pelvic cavity; (2) masses without an apparent organ-based primary site; (3) mild to moderate homogeneous or heterogeneous enhancement in solid area on enhanced CT; and (4) secondary manifestations, such as ascites, hepatic metastases, lymphadenopathy, hydronephrosis and hydroureter. The prognosis and overall survival rates were generally poor. Commonly used treatment strategies including aggressive tumor resection, polychemotherapy, and radiotherapy, showed various therapeutic effects. CT of DSRCT shows characteristic features that are helpful in diagnosis. Early discovery and complete resection, coupled with postoperative adjuvant chemotherapy, are important for prognosis of DSRCT. Whole abdominopelvic rather than locoregional radiotherapy is more effective for unresectable DSRCT.
doi:10.3748/wjg.v20.i17.5157
PMCID: PMC4009557  PMID: 24803835
Desmoplastic small round cell tumor; Peritoneum; Pathology; Computed tomography; Clinical features
11.  The Immunosuppressant Protosappanin A Diminished Recipient T Cell Migration into Allograft via Inhibition of IP-10 in Rat Heart Transplant 
PLoS ONE  2014;9(5):e96138.
The immunosuppressant Protosappanin A (PrA), isolated from the medicinal herb, promotes cardiac allograft survival, diminishes inflammatory cell infiltration, and inhibits interferon γ-induced protein 10 kDa (IP-10) mRNA expression in rats cardiac grafts. Binding of the chemokine IP-10 to its cognate receptor, CXCR3, plays crucial roles in allograft immunity, especially by mediating the recruitment of effector T cells to allografted tissues. In this study, we attempted to determine whether PrA-mediated inhibition of IP-10 contributes to the effect of reduced T cell infiltration into cardiac allograft within a rat model. Administration of PrA (25 mg/kg daily) via oral gavage following heart transplantation significantly reduced the increase of IP-10 mRNA level in allograft and prevented IP-10 secretion by peripheral blood mononuclear cells (PBMC) isolated from recipient rats seven days posttransplantation. Furthermore, in vitro experiments demonstrated that PrA addition to control PBMC prevented IP-10 secretion. Chemotactic migration assays were utilized to evaluate recipient T cell migration towards PBMC supernatant. PrA administration impaired PBMC supernatant-induced T cell migration. Additional in vitro experiments revealed that PrA slightly reduced naïve T cell migration towards chemokines. The presence of IP-10 in PBMC supernatant prevented PrA from reducing T cell migration in PrA-treated recipients. Neither CXCR3 chemokine ligand Mig nor non-CXCR3 chemokine ligand SDF-1 had any effect on T cell migration in PrA-treated recipients. The addition of anti-CXCR3 antibody restored PrA-mediated inhibition of T cell migration. Immunofluorescence microscopy showed that IP-10 was expressed mainly in CD68 positive infiltrating monocytes. Furthermore, PrA consistently reduced CXCR3+T cell infiltration into cardiac allografts. The reduced intensity of CXCR3 staining in PrA-treated allografts contributed to the previously depressed naïve T cell migrating activity induced by PrA. Collectively, these data indicate that PrA inhibition of IP-10 activity reduced recipient T cell migration and infiltration of cardiac allografts, thus partially explaining the immunosuppressive effect of PrA.
doi:10.1371/journal.pone.0096138
PMCID: PMC4010525  PMID: 24798458
12.  Exomic Analysis of Myxoid Liposarcomas, Synovial Sarcomas, and Osteosarcomas 
Genes, chromosomes & cancer  2013;53(1):15-24.
Bone and soft tissue sarcomas are a group of histologically heterogeneous and relatively uncommon tumors. To explore their genetic origins, we sequenced the exomes of 13 osteosarcomas, eight myxoid liposarcomas (MLPS), and seven synovial sarcomas (SYN). These tumors had few genetic alterations (median of 10.8). Nevertheless, clear examples of driver gene mutations were observed, including canonical mutations in TP53, PIK3CA, SETD2, AKT1, and subclonal mutation in FBXW7. Of particular interest were mutations in H3F3A, encoding the variant histone H3.3. Mutations in this gene have only been previously observed in gliomas. Loss of heterozygosity of exomic regions was extensive in osteosarcomas but rare in SYN and MLPS. These results provide intriguing nucleotide-level information on these relatively uncommon neoplasms and highlight pathways that help explain their pathogenesis.
doi:10.1002/gcc.22114
PMCID: PMC4007004  PMID: 24190505
13.  Genetic Polymorphism of Interferon Regulatory Factor 5 (IRF5) Correlates with Allograft Acute Rejection of Liver Transplantation 
PLoS ONE  2014;9(4):e94426.
Background
Although liver transplantation is one of the most efficient curative therapies of end stage liver diseases, recipients may suffer liver graft loss opst-operation. IRF-5, a member of Interferon Regulatory Factors, functions as a key regulator in TLR4 cascade, and is capable of inducing inflammatory cytokines. Although TLR4 has been proved to contribute to acute allograft rejection, including after liver transplantation, the correlation between IRF5 gene and acute rejection has not been elucidated yet.
Methods
The study enrolled a total of 289 recipients, including 39 females and 250 males, and 39 recipients developed acute allograft rejection within 6 months post-transplantation. The allograft rejections were diagnosed by liver biopsies. Genome DNA of recipients was extracted from pre-operative peripheral blood. Genotyping of IRF-5, including rs3757385, rs752637 and rs11761199, was performed, followed by SNP frequency and Hardy-Weinberg equilibrium analysis.
Results
The genetic polymorphism of rs3757385 was found associated with acute rejection. G/G homozygous individuals were at higher risk of acute rejection, with a P value of 0.042 (OR = 2.34 (1.07–5.10)).
Conclusions
IRF5, which transcriptionally activates inflammatory cytokines, is genetically associated with acute rejection and might function as a risk factor for acute rejection of liver transplantations.
doi:10.1371/journal.pone.0094426
PMCID: PMC4005731  PMID: 24788560
14.  Multiple-Site Hemodynamic Analysis of Doppler Ultrasound with an Adaptive Color Relation Classifier for Arteriovenous Access Occlusion Evaluation 
The Scientific World Journal  2014;2014:203148.
This study proposes multiple-site hemodynamic analysis of Doppler ultrasound with an adaptive color relation classifier for arteriovenous access occlusion evaluation in routine examinations. The hemodynamic analysis is used to express the properties of blood flow through a vital access or a tube, using dimensionless numbers. An acoustic measurement is carried out to detect the peak-systolic and peak-diastolic velocities of blood flow from the arterial anastomosis sites (A) to the venous anastomosis sites (V). The ratio of the supracritical Reynolds (Resupra) number and the resistive (Res) index quantitates the degrees of stenosis (DOS) at multiple measurement sites. Then, an adaptive color relation classifier is designed as a nonlinear estimate model to survey the occlusion level in monthly examinations. For 30 long-term follow-up patients, the experimental results show the proposed screening model efficiently evaluates access occlusion.
doi:10.1155/2014/203148
PMCID: PMC4032682  PMID: 24892039
15.  Numb Promotes Cell Proliferation and Correlates with Poor Prognosis in Hepatocellular Carcinoma 
PLoS ONE  2014;9(4):e95849.
Background
Numb is an evolutionary conserved protein that plays critical roles in cell fate determination, cell adhesion, cell migration and a number of signaling pathways, but evidence for a substantial involvement of Numb in HCC has remained unclear. The present study was aimed to investigate the clinical and prognostic significance of Numb and its role in hepatocellular carcinoma (HCC).
Methodology
The expression of Numb was detected in 107 cases of clinical paraffin-embedded hepatocellular carcinoma tissues,5 matched paris of fresh tissues and six hepatocellular cell lines by immunohistochemistry with clinicopathological analyses,RT-PCR or Western blot. Moreover, loss of function and gain of function assays were performed to evaluate the effect of Numb on cell proliferation in vitro.
Conclusions
We found that Numb was obviously up-regulated in HCC tissues and cell lines (p<0.05). The Numb up-regulation correlated significantly with poor prognosis, and Numb status was identified as an independent prognostic factor. Over-expression of Numb increased proliferation in SMMC-7721 and BEL-7402 cells, while knock-down of Numb showed the opposite effect. Our study indicates that Numb up-regulation significantly correlates with cell proliferation and poor prognosis in hepatocellular carcinoma patients. It may be a useful biomarker for therapeutic strategy in hepatocellular carcinoma treatment.
doi:10.1371/journal.pone.0095849
PMCID: PMC4000222  PMID: 24770339
16.  Implementing Fluorescence Anisotropy Screening and Crystallographic Analysis to Define PKA Isoform-Selective Activation by cAMP Analogs 
ACS chemical biology  2013;8(10):10.1021/cb400247t.
Cyclic AMP (cAMP) is a ubiquitous second messenger that regulates many proteins, most notably cAMP-dependent protein kinase (PKA). PKA holoenzymes (comprised of two catalytic (C) and two regulatory (R) subunits) regulate a wide variety of cellular processes, and its functional diversity is amplified by the presence of four R-subunit isoforms, RIα, RIβ, RIIα, and RIIβ. Although these isoforms all respond to cAMP, they are functionally non-redundant and exhibit different biochemical properties. In order to understand the functional differences between these isoforms, we screened cAMP derivatives for their ability to selectively activate RI and RII PKA holoenzymes using a fluorescence anisotropy assay. Our results indicate that RIα holoenzymes are selectively activated by C8-substituted analogs and RIIβ holoenzymes by N6-substituted analogs, where HE33 is the most prominent RII activator. We also solved the crystal structures of both RIα and RIIβ bound to HE33. The RIIβ structure shows the bulky aliphatic substituent of HE33 is fully encompassed by a pocket comprising of hydrophobic residues. RIα lacks this hydrophobic lining in Domain A and the side chains are displaced to accommodate the HE33 di-propyl groups. Comparison between cAMP-bound structures reveals that RIIβ, but not RIα, contains a cavity near the N6 site. This study suggests that the selective activation of RII over RI isoforms by N6 analogs is driven by the spatial and chemical constraints of Domain A and paves the way for the development of potent non-cyclic nucleotides activators to specifically target PKA iso-holoenyzmes.
doi:10.1021/cb400247t
PMCID: PMC3827627  PMID: 23978166
cAMP-dependent protein kinase; cyclic nucleotide analogs; isoform selectivity; fluorescence anisotropy; x-ray crystallography
17.  2-[2,6-Bis(pyrazin-2-yl)pyridin-4-yl]benzoic acid 
In the title compound, C20H13N5O2, the two pyrazine rings are nearly coplanar with the central pyridine ring, forming dihedral angles of 2.21 (9) and 4.57 (9)°. In contrast, the strong steric hindrance caused by the ortho-carboxyl group on the phenyl ring makes this ring rotate out of the attached pyridine ring plane by 52.60 (9)°. The carboxyl group is twisted from the phenyl ring by 22.6 (1)°. In the crystal, aromatic π–π stacking inter­actions [centroid–centroid distances = 3.9186 (4) and 3.9794 (5) Å] occur between the anti­parallel mol­ecules, generating infinite chains along [100]. O—H⋯O hydrogen bonds connect the chains, leading to the formation of a two-dimensional supra­molecular network parallel to (010). Inter­molecular C—H⋯N hydrogen bonds are also observed.
doi:10.1107/S1600536814008496
PMCID: PMC4011275  PMID: 24860380
18.  Exosomes from Drug-Resistant Breast Cancer Cells Transmit Chemoresistance by a Horizontal Transfer of MicroRNAs 
PLoS ONE  2014;9(4):e95240.
Adriamycin and docetaxel are two agents commonly used in treatment of breast cancer, but their efficacy is often limited by the emergence of chemoresistance. Recent studies indicate that exosomes act as vehicles for exchange of genetic cargo between heterogeneous populations of tumor cells, engendering a transmitted drug resistance for cancer development and progression. However, the specific contribution of breast cancer-derived exosomes is poorly understood. Here we reinforced other's report that human breast cancer cell line MCF-7/S could acquire increased survival potential from its resistant variants MCF-7/Adr and MCF-7/Doc. Additionally, exosomes of the latter, A/exo and D/exo, significantly modulated the cell cycle distribution and drug-induced apoptosis with respect to S/exo. Exosomes pre-treated with RNase were unable to regulate cell cycle and apoptosis resistance, suggesting an RNA-dependent manner. Microarray and polymerase chain reaction for the miRNA expression profiles of A/exo, D/exo, and S/exo demonstrated that they loaded selective miRNA patterns. Following A/exo and D/exo transfer to recipient MCF-7/S, the same miRNAs were significantly increased in acquired cells. Target gene prediction and pathway analysis showed the involvement of miR-100, miR-222, and miR-30a in pathways implicated in cancer pathogenesis, membrane vesiculation and therapy failure. Furthermore, D/exo co-culture assays and miRNA mimics transfection experiments indicated that miR-222-rich D/exo could alter target gene expression in MCF-7/S. Our results suggest that drug-resistant breast cancer cells may spread resistance capacity to sensitive ones by releasing exosomes and that such effects could be partly attributed to the intercellular transfer of specific miRNAs.
doi:10.1371/journal.pone.0095240
PMCID: PMC3989268  PMID: 24740415
19.  The formins Cdc12 and For3 cooperate during contractile ring assembly in cytokinesis 
The Journal of Cell Biology  2013;203(1):101-114.
Multiple formins cooperate during cytokinesis, but their functions in de novo actin assembly at the division site play the primary role in contractile ring assembly.
Both de novo–assembled actin filaments at the division site and existing filaments recruited by directional cortical transport contribute to contractile ring formation during cytokinesis. However, it is unknown which source is more important. Here, we show that fission yeast formin For3 is responsible for node condensation into clumps in the absence of formin Cdc12. For3 localization at the division site depended on the F-BAR protein Cdc15, and for3 deletion was synthetic lethal with mutations that cause defects in contractile ring formation. For3 became essential in cells expressing N-terminal truncations of Cdc12, which were more active in actin assembly but depended on actin filaments for localization to the division site. In tetrad fluorescence microscopy, double mutants of for3 deletion and cdc12 truncations were severely defective in contractile ring assembly and constriction, although cortical transport of actin filaments was normal. Together, these data indicate that different formins cooperate in cytokinesis and that de novo actin assembly at the division site is predominant for contractile ring formation.
doi:10.1083/jcb.201305022
PMCID: PMC3798249  PMID: 24127216
20.  Identification of Thyroid Carcinoma Related Genes with mRMR and Shortest Path Approaches 
PLoS ONE  2014;9(4):e94022.
Thyroid cancer is a malignant neoplasm originated from thyroid cells. It can be classified into papillary carcinomas (PTCs) and anaplastic carcinomas (ATCs). Although ATCs are in an very aggressive status and cause more death than PTCs, their difference is poorly understood at molecular level. In this study, we focus on the transcriptome difference among PTCs, ATCs and normal tissue from a published dataset including 45 normal tissues, 49 PTCs and 11 ATCs, by applying a machine learning method, maximum relevance minimum redundancy, and identified 9 genes (BCL2, MRPS31, ID4, RASAL2, DLG2, MY01B, ZBTB5, PRKCQ and PPP6C) and 1 miscRNA (miscellaneous RNA, LOC646736) as important candidates involved in the progression of thyroid cancer. We further identified the protein-protein interaction (PPI) sub network from the shortest paths among the 9 genes in a PPI network constructed based on STRING database. Our results may provide insights to the molecular mechanism of the progression of thyroid cancer.
doi:10.1371/journal.pone.0094022
PMCID: PMC3981740  PMID: 24718460
21.  Src Is Required for Mechanical Stretch-Induced Cardiomyocyte Hypertrophy through Angiotensin II Type 1 Receptor-Dependent β-Arrestin2 Pathways 
PLoS ONE  2014;9(4):e92926.
Angiotensin II (AngII) type 1 receptor (AT1-R) can be activated by mechanical stress (MS) without the involvement of AngII during the development of cardiomyocyte hypertrophy, in which G protein-independent pathways are critically involved. Although β-arrestin2-biased signaling has been speculated, little is known about how AT1-R/β-arrestin2 leads to ERK1/2 activation. Here, we present a novel mechanism by which Src kinase mediates AT1-R/β-arrestin2-dependent ERK1/2 phosphorylation in response to MS. Differing from stimulation by AngII, MS-triggered ERK1/2 phosphorylation is neither suppressed by overexpression of RGS4 (the negative regulator of the G-protein coupling signal) nor by inhibition of Gαq downstream protein kinase C (PKC) with GF109203X. The release of inositol 1,4,5-triphosphate (IP3) is increased by AngII but not by MS. These results collectively suggest that MS-induced ERK1/2 activation through AT1-R might be independent of G-protein coupling. Moreover, either knockdown of β-arrestin2 or overexpression of a dominant negative mutant of β-arrestin2 prevents MS-induced activation of ERK1/2. We further identifies a relationship between Src, a non-receptor tyrosine kinase and β-arrestin2 using analyses of co-immunoprecipitation and immunofluorescence after MS stimulation. Furthermore, MS-, but not AngII-induced ERK1/2 phosphorylation is attenuated by Src inhibition, which also significantly improves pressure overload-induced cardiac hypertrophy and dysfunction in mice lacking AngII. Finally, MS-induced Src activation and hypertrophic response are abolished by candesartan but not by valsartan whereas AngII-induced responses can be abrogated by both blockers. Our results suggest that Src plays a critical role in MS-induced cardiomyocyte hypertrophy through β-arrestin2-associated angiotensin II type 1 receptor signaling.
doi:10.1371/journal.pone.0092926
PMCID: PMC3974699  PMID: 24699426
22.  Diagnostic Value of Virtual Touch Tissue Quantification for Breast Lesions with Different Size 
BioMed Research International  2014;2014:142504.
Purpose. To evaluate diagnostic value of the virtual touch tissue quantification (VTTQ) for breast lesions with different sizes. Materials and Methods. Patients with 206 breast lesions were categorized into three groups according to lesion size (<10 mm, 10–20 mm, and >20 mm). Breast lesions were examined by conventional ultrasound and VTTQ, and shear wave velocity (SWV) of each lesion and adjacent normal breast tissue were measured. Diagnoses were confirmed by pathological examination after surgery. The receiver-operating characteristic curve (ROC) analyses were performed to evaluate the diagnostic value of SWV, and the area under curves (AUC) was compared among groups. Results. SWV of malignant lesions was much higher than that of benign lesions, whereas the difference was not obvious for lesions <10 mm (P = 0.15). There was statistical significant difference of AUC between lesions <10 mm and 10–20 mm (P < 0.05), as well as lesions <10 mm and >20 mm (P < 0.05). The sensitivity of lesions <10 mm was 33.33%, which was relatively low compared to other groups. Conclusion. According to our results, VTTQ is a promising method for breast lesions >10 mm, and further studies were warranted to improve sensitivity of VTTQ for breast lesions <10 mm.
doi:10.1155/2014/142504
PMCID: PMC3996890  PMID: 24800205
23.  β-catenin is overexpressed in hepatic fibrosis and blockage of Wnt/β-catenin signaling inhibits hepatic stellate cell activation 
Molecular Medicine Reports  2014;9(6):2145-2151.
β-catenin, a core component of Wnt/β-catenin signaling, has been shown to be an important regulator of cellular proliferation and differentiation. Abnormal activation of Wnt/β-catenin signaling promotes tissue fibrogenesis. In the present study, the role of β-catenin during liver fibrogenesis was analyzed and the functional effects of β-catenin gene silencing in hepatic stellate cells (HSCs) using small interfering (si)RNA were investigated. The expression of β-catenin in human hepatic fibrosis tissues of different grades and normal human hepatic tissues was examined using immunohistochemistry. To inhibit the Wnt/β-catenin signaling pathway, siRNA for β-catenin was developed and transiently transfected into HSC-T6 cells using Lipofectamine 2000. β-catenin expression was evaluated by quantitative polymerase chain reaction (qPCR) and western blot analysis. The expression of collagen types I and III was evaluated by qPCR and immunofluorescent staining. Cellular proliferation and the cell cycle were analyzed using a methyl thiazolyl tetrazolium assay. Apoptosis was assessed by Annexin V staining. A higher expression level of β-catenin was identified in the patients with high-grade hepatic fibrosis in comparison with that of the normal controls. Additionally, β-catenin siRNA molecules were successfully transfected into HSCs and induced inhibition of β-catenin expression in a time-dependent manner. β-catenin siRNA treatment also inhibited synthesis of collagen types I and I in transfected HSCs. Furthermore, compared with those of the control group, siRNA-mediated knockdown of β-catenin in HSC-T6 cells inhibited cell proliferation and resulted in cell apoptosis. This study suggests a significant functional role for β-catenin in the development of liver fibrosis and demonstrates that downregulation of the Wnt/β-catenin signaling pathway inhibits HSC activation. Thus, this study provides a novel strategy for the treatment of hepatic fibrosis.
doi:10.3892/mmr.2014.2099
PMCID: PMC4055486  PMID: 24691643
hepatic fibrosis; β-catenin; hepatic stellate cells; RNA interference
24.  The Stratifying Value of Hangzhou Criteria in Liver Transplantation for Hepatocellular Carcinoma 
PLoS ONE  2014;9(3):e93128.
Background/Aims
The selection criteria for patients with hepatocellular carcinoma (HCC) as candidates for deceased donor liver transplantation (DDLT) are well studied. In this era of limited deceased donor organs, the value of living donor liver transplantation (LDLT) for HCC remains controversial. The aim of the present study was to verify the stratification value of the Hangzhou criteria for LDLT.
Methods
The data of 47 LDLT recipients and 94 matched DDLT recipients at our center were evaluated. Overall survival and tumor-free survival were calculated. Prognostic factors influencing post-liver transplantation (LT) survival were identified. The stratification values of the Hangzhou criteria and Milan criteria were compared.
Results
LDLT recipients spent much less time on the waiting list. The post-LT survival of recipients fulfilling the Milan criteria and recipients fulfilling the Hangzhou criteria were comparable (P>0.05). The overall and tumor-free survival did not differ statistically between the two groups. In both groups, more recipients not meeting the Milan criteria but with a satisfactory outcome were identified by the Hangzhou criteria. Among recipients who did not meet the Hangzhou criteria, tumor-free survival was better for the LDLT recipients than the DDLT recipients (P = 0.024).
Conclusions
The Hangzhou criteria are reliable for stratifying HCC patients in terms of prognosis. HCC patients fulfilling the Hangzhou criteria gain satisfactory survival from LT. Outcomes after LDLT are better than those after DDLT for HCC patients who do not meet the Hangzhou criteria.
doi:10.1371/journal.pone.0093128
PMCID: PMC3968059  PMID: 24676010
25.  Pulmonary lymphangitic carcinomatosis in liver carcinoma: a rare case report and literature review 
Primary liver carcinoma is the most important malignant disease. The nodular metastatic foci of liver carcinoma are usually found in the lung, adrenal gland or abdomen after resection or transplantation. Pulmonary lymphangitic carcinomatosis (PLC) accounts for approximately 6% to 8% of metastatic cancer in the lung. The occurrence of PLC is extremely rare in liver carcinoma. Herein we report the case of a patient with PLC after liver transplantation due to liver carcinoma. PLC was confirmed by clinical manifestations, imaging studies and cytologic examination of exfoliated cells in the pleural effusion.
doi:10.1186/1477-7819-12-66
PMCID: PMC3986917  PMID: 24669948
Liver carcinoma; Liver transplantation; Metastasis; Pulmonary lymphangitic carcinomatosis

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