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1.  Traditional Chinese Herb Combined with Surgery versus Surgery for Varicocele Infertility: A Systematic Review and Meta-Analysis 
Objective. The objective of this study was to conduct a systematic review to assess the effectiveness and safety of traditional Chinese herb combined with surgery for male varicocele infertility compared to surgery. Methods. Randomized controlled trials (RCTs) data of traditional Chinese herbs combined with surgery for male varicocele fertility versus surgery were collected by searching the Cochrane Library, Embase, PubMed, and Chinese databases. The risk of bias was assessed using Cochrane Handbook. Study outcomes were presented as risk ratios (RRs) for dichotomous data. Results. Seventeen of 72 potentially relevant trials met the inclusion criteria. The methodological qualities of the RCTs were low. Compared with the surgery group, the traditional Chinese herb combined with surgery group had superiority in pregnancy rate at 3-month (RR = 1.76, and P = 0.008), 6-month (RR = 1.58, and P = 0.0005), and 2-year (RR = 1.58, and P = 0.0005) follow-ups. No RCT was found to describe the side effects. Conclusion. On considering the low methodological quality of RCTs, there was no enough evidence on traditional Chinese herb with surgery for male varicocele infertility, and more high-quality RCTs of large sample sizes are required.
PMCID: PMC4325216
2.  Molecular insights into the interaction of the ribosomal stalk protein with elongation factor 1α 
Nucleic Acids Research  2014;42(22):14042-14052.
In all organisms, the large ribosomal subunit contains multiple copies of a flexible protein, the so-called ‘stalk’. The C-terminal domain (CTD) of the stalk interacts directly with the translational GTPase factors, and this interaction is required for factor-dependent activity on the ribosome. Here we have determined the structure of a complex of the CTD of the archaeal stalk protein aP1 and the GDP-bound archaeal elongation factor aEF1α at 2.3 Å resolution. The structure showed that the CTD of aP1 formed a long extended α-helix, which bound to a cleft between domains 1 and 3 of aEF1α, and bridged these domains. This binding between the CTD of aP1 and the aEF1α•GDP complex was formed mainly by hydrophobic interactions. The docking analysis showed that the CTD of aP1 can bind to aEF1α•GDP located on the ribosome. An additional biochemical assay demonstrated that the CTD of aP1 also bound to the aEF1α•GTP•aminoacyl-tRNA complex. These results suggest that the CTD of aP1 interacts with aEF1α at various stages in translation. Furthermore, phylogenetic perspectives and functional analyses suggested that the eukaryotic stalk protein also interacts directly with domains 1 and 3 of eEF1α, in a manner similar to the interaction of archaeal aP1 with aEF1α.
PMCID: PMC4267659  PMID: 25428348
3.  Reaction pathways and free energy profiles for spontaneous hydrolysis of urea and tetramethylurea: Unexpected substituent effects 
Organic & biomolecular chemistry  2013;11(43):7595-7605.
It has been difficult to directly measure the spontaneous hydrolysis rate of urea and, thus, 1,1,3,3-tetramethylurea (Me4U) was used as a model to determine the “experimental” rate constant for urea hydrolysis. The use of Me4U was based on an assumption that the rate of urea hydrolysis should be 2.8 times that of Me4U hydrolysis because the rate of acetamide hydrolysis is 2.8 times that of N,N-dimethyl-acetamide hydrolysis. The present first-principles electronic-structure calculations on the competing non-enzymatic hydrolysis pathways have demonstrated that the dominant pathway is the neutral hydrolysis via the CN addition for both urea (when pH<~11.6) and Me4U (regardless of pH), unlike the non-enzymatic hydrolysis of amides where alkaline hydrolysis is dominant. Based on the computational data, the substituent shift of free energy barrier calculated for the neutral hydrolysis is remarkably different from that for the alkaline hydrolysis, and the rate constant for the urea hydrolysis should be ~1.3×109-fold lower than that (4.2×10−12 s−1) measured for the Me4U hydrolysis. As a result, the rate enhancement and catalytic proficiency of urease should be 1.2×1025 and 3×1027 M−1, respectively, suggesting that urease surpasses proteases and all other enzymes in its power to enhance the rate of reaction. All of the computational results are consistent with available experimental data for Me4U, suggesting that the computational prediction for urea is reliable.
PMCID: PMC3870011  PMID: 24097048
4.  Rice Stripe Tenuivirus NSvc2 Glycoproteins Targeted to the Golgi Body by the N-Terminal Transmembrane Domain and Adjacent Cytosolic 24 Amino Acids via the COP I- and COP II-Dependent Secretion Pathway 
Journal of Virology  2014;88(6):3223-3234.
The NSvc2 glycoproteins encoded by Rice stripe tenuivirus (RSV) share many characteristics common to the glycoproteins found among Bunyaviridae. Within this viral family, glycoproteins targeting to the Golgi apparatus play a pivotal role in the maturation of the enveloped spherical particles. RSV particles, however, adopt a long filamentous morphology. Recently, RSV NSvc2 glycoproteins were shown to localize exclusively to the ER in Sf9 insect cells. Here, we demonstrate that the amino-terminal NSvc2 (NSvc2-N) targets to the Golgi apparatus in Nicotiana benthamiana cells, whereas the carboxyl-terminal NSvc2 (NSvc2-C) accumulates in the endoplasmic reticulum (ER). Upon coexpression, NSvc2-N redirects NSvc2-C from the ER to the Golgi bodies. The NSvc2 glycoproteins move together with the Golgi stacks along the ER/actin network. The targeting of the NSvc2 glycoproteins to the Golgi bodies was strictly dependent on functional anterograde traffic out of the ER to the Golgi bodies or on a retrograde transport route from the Golgi apparatus. The analysis of truncated and chimeric NSvc2 proteins demonstrates that the Golgi targeting signal comprises amino acids 269 to 315 of NSvc2-N, encompassing the transmembrane domain and 24 adjacent amino acids in the cytosolic tail. Our findings demonstrate for the first time that the glycoproteins from an unenveloped Tenuivirus could target Golgi bodies in plant cells.
IMPORTANCE NSvc2 glycoprotein encoded by unenveloped Rice stripe tenuivirus (RSV) share many characteristics in common with glycoprotein found among Bunyaviridae in which all members have membrane-enveloped sphere particle. Recently, RSV NSvc2 glycoproteins were shown to localize exclusively to the ER in Sf9 insect cells. In this study, we demonstrated that the RSV glycoproteins could target Golgi bodies in plant cells. The targeting of NSvc2 glycoproteins to the Golgi bodies was dependent on active COP II or COP I. The Golgi targeting signal was mapped to the 23-amino-acid transmembrane domain and the adjacent 24 amino acids of the cytosolic tail of the NSvc2-N. In light of the evidence from viruses in Bunyaviridae that targeting Golgi bodies is important for the viral particle assembly and vector transmission, we propose that targeting of RSV glycoproteins into Golgi bodies in plant cells represents a physiologically relevant mechanism in the maturation of RSV particle complex for insect vector transmission.
PMCID: PMC3957912  PMID: 24390331
5.  NIM: A Node Influence Based Method for Cancer Classification 
The classification of different cancer types owns great significance in the medical field. However, the great majority of existing cancer classification methods are clinical-based and have relatively weak diagnostic ability. With the rapid development of gene expression technology, it is able to classify different kinds of cancers using DNA microarray. Our main idea is to confront the problem of cancer classification using gene expression data from a graph-based view. Based on a new node influence model we proposed, this paper presents a novel high accuracy method for cancer classification, which is composed of four parts: the first is to calculate the similarity matrix of all samples, the second is to compute the node influence of training samples, the third is to obtain the similarity between every test sample and each class using weighted sum of node influence and similarity matrix, and the last is to classify each test sample based on its similarity between every class. The data sets used in our experiments are breast cancer, central nervous system, colon tumor, prostate cancer, acute lymphoblastic leukemia, and lung cancer. experimental results showed that our node influence based method (NIM) is more efficient and robust than the support vector machine, K-nearest neighbor, C4.5, naive Bayes, and CART.
PMCID: PMC4144086  PMID: 25180045
6.  Priming cancer cells for drug resistance: role of the fibroblast niche 
Frontiers in biology  2014;9(2):114-126.
Conventional and targeted chemotherapies remain integral strategies to treat solid tumors. Despite the large number of anti-cancer drugs available, chemotherapy does not completely eradicate disease. Disease recurrence and the growth of drug resistant tumors remain significant problems in anti-cancer treatment. To develop more effective treatment strategies, it is important to understand the underlying cellular and molecular mechanisms of drug resistance. It is generally accepted that cancer cells do not function alone, but evolve through interactions with the surrounding tumor microenvironment. As key cellular components of the tumor microenvironment, fibroblasts regulate the growth and progression of many solid tumors. Emerging studies demonstrate that fibroblasts secrete a multitude of factors that enable cancer cells to become drug resistant. This review will explore how fibroblast secretion of soluble factors act on cancer cells to enhance cancer cell survival and cancer stem cell renewal, contributing to the development of drug resistant cancer.
PMCID: PMC4101896  PMID: 25045348
fibroblasts; tumor recurrence; drug resistance; cell survival; stem cells; tumor dormancy
7.  Application of fluorodeoxyglucose positron emission tomography in the management of head and neck cancers 
World Journal of Radiology  2014;6(6):238-251.
The use of fluorodeoxyglucose positron emission tomography (FDG PET) scan technology in the management of head and neck cancers continues to increase. We discuss the biology of FDG uptake in malignant lesions and also discuss the physics of PET imaging. The various parameters described to quantify FDG uptake in cancers including standardized uptake value, metabolic tumor volume and total lesion glycolysis are presented. PET scans have found a significant role in the diagnosis and staging of head and neck cancers. They are also being increasingly used in radiation therapy treatment planning. Many groups have also used PET derived values to serve as prognostic indicators of outcomes including loco-regional control and overall survival. FDG PET scans are also proving very useful in assessing the efficacy of treatment and management and follow-up of head and neck cancer patients. This review article focuses on the role of FDG-PET computed tomography scans in these areas for squamous cell carcinoma of the head and neck. We present the current state of the art and speculate on the future applications of this technology including protocol development, newer imaging methods such as combined magnetic resonance and PET imaging and novel radiopharmaceuticals that can be used to further study tumor biology.
PMCID: PMC4072811  PMID: 24976927
Fluorodeoxyglucose; Positron emission tomography; Squamous cell carcinoma; Head and neck cancer; Radiation therapy planning
8.  Directed Evolution and Structural Analysis of NADPH-Dependent Acetoacetyl Coenzyme A (Acetoacetyl-CoA) Reductase from Ralstonia eutropha Reveals Two Mutations Responsible for Enhanced Kinetics 
Applied and Environmental Microbiology  2013;79(19):6134-6139.
NADPH-dependent acetoacetyl-coenzyme A (acetoacetyl-CoA) reductase (PhaB) is a key enzyme in the synthesis of poly(3-hydroxybutyrate) [P(3HB)], along with β-ketothiolase (PhaA) and polyhydroxyalkanoate synthase (PhaC). In this study, PhaB from Ralstonia eutropha was engineered by means of directed evolution consisting of an error-prone PCR-mediated mutagenesis and a P(3HB) accumulation-based in vivo screening system using Escherichia coli. From approximately 20,000 mutants, we obtained two mutant candidates bearing Gln47Leu (Q47L) and Thr173Ser (T173S) substitutions. The mutants exhibited kcat values that were 2.4-fold and 3.5-fold higher than that of the wild-type enzyme, respectively. In fact, the PhaB mutants did exhibit enhanced activity and P(3HB) accumulation when expressed in recombinant Corynebacterium glutamicum. Comparative three-dimensional structural analysis of wild-type PhaB and highly active PhaB mutants revealed that the beneficial mutations affected the flexibility around the active site, which in turn played an important role in substrate recognition. Furthermore, both the kinetic analysis and crystal structure data supported the conclusion that PhaB forms a ternary complex with NADPH and acetoacetyl-CoA. These results suggest that the mutations affected the interaction with substrates, resulting in the acquirement of enhanced activity.
PMCID: PMC3811355  PMID: 23913421
9.  Genetic Algorithm and Graph Theory Based Matrix Factorization Method for Online Friend Recommendation 
The Scientific World Journal  2014;2014:162148.
Online friend recommendation is a fast developing topic in web mining. In this paper, we used SVD matrix factorization to model user and item feature vector and used stochastic gradient descent to amend parameter and improve accuracy. To tackle cold start problem and data sparsity, we used KNN model to influence user feature vector. At the same time, we used graph theory to partition communities with fairly low time and space complexity. What is more, matrix factorization can combine online and offline recommendation. Experiments showed that the hybrid recommendation algorithm is able to recommend online friends with good accuracy.
PMCID: PMC3976802  PMID: 24757410
10.  Regression of vascular calcification following an acute episode of calciphylaxis: a case report 
In clinical situations, vascular calcification tends to progress and is difficult to completely arrest or reverse. Calciphylaxis, a severe complication of end-stage renal disease, is a specific form of vascular calcification. Control studies have provided evidence that monotherapy with sodium thiosulfate or cinacalcet delays the progression of vascular calcification. Successful treatment of calciphylaxis with sodium thiosulfate or cinacalcet has also been reported. We report a case demonstrating the regression of vascular calcification following an acute episode of necrotic skin lesions suspected to be calciphylaxis. During the successful multimodal treatment, sodium thiosulfate and cinacalcet were sequentially administered in addition to surgical debridement and percutaneous transluminal angioplasty.
Case presentation
We describe the case of a 71-year-old Asian woman on hemodialysis who presented with suspected calciphylaxis lesions in her lower left leg. Plain radiographs revealed diffuse calcified vessel changes in her lower extremities. During the initial wound treatment with a course of intravenous sodium thiosulfate, our patient’s predialysis serum levels of total calcium markedly increased, yielding no calciphylaxis improvement. The necrotic wounds began healing only after surgical debridement. A percutaneous transluminal angioplasty was performed to dilate a 70% stenosis in her left posterior tibial artery. Our patient was then treated with cinacalcet, resulting in improved control of her calcium, phosphate and parathyroid hormone serum levels. The lesions completely healed after six months of multimodal treatment. Repeated plain radiographs in the following two years revealed gradual vascular calcification regression in her lower extremities.
In addition to the favorable outcome of our patient’s wounds, radiology was used to document the regression of calcification in the large and small arteries of her lower limbs. However, it is difficult to determine the precise mechanism of the multimodal treatment that caused the vascular calcification regression and wound healing. The clinical course suggested that the surgical treatment and percutaneous transluminal angioplasty substantially contributed to healing her wounds. Cinacalcet and sodium thiosulfate may have played distinct roles in the regression of her vascular calcification. A well-controlled study or large case series are required to assess the additive effects of these agents when treating vascular calcification.
PMCID: PMC3930056  PMID: 24524553
Calciphylaxis; Cinacalcet; Sodium thiosulfate; Vascular calcification
11.  NADPH Oxidase 1 and Its Derived Reactive Oxygen Species Mediated Tissue Injury and Repair 
Reactive oxygen species are mostly viewed to cause oxidative damage to various cells and induce organ dysfunction after ischemia-reperfusion injury. However, they are also considered as crucial molecules for cellular signal transduction in biology. NADPH oxidase, whose only function is reactive oxygen species production, has been extensively investigated in many cell types especially phagocytes. The deficiency of NADPH oxidase extends the process of inflammation and delays tissue repair, which causes chronic granulomatous disease in patients. NADPH oxidase 1, one member of the NADPH oxidase family, is not only constitutively expressed in a variety of tissues, but also induced to increase expression in both mRNA and protein levels under many circumstances. NADPH oxidase 1 and its derived reactive oxygen species are suggested to be able to regulate inflammation reaction, cell proliferation and migration, and extracellular matrix synthesis, which contribute to the processes of tissue injury and repair.
PMCID: PMC3942082  PMID: 24669283
12.  Endoplasmic reticulum stress-induced PCD and caspase-like activities involved 
Plant cells, like cells from other kingdoms, have the ability to self-destruct in a genetically controlled manner. This process is defined as Programmed cell death (PCD). PCD can be triggered by various stimuli in plants including by endoplasmic reticulum (ER) stress. Research in the past two decades discovered that disruption of protein homeostasis in the ER could cause ER stress, which when prolonged/unresolved leads cells into PCD. ER stress-induced PCD is part of several plant processes, for instance, drought and heat stress have been found to elicit ER stress-induced PCD. Despite the importance of ER stress-induced PCD in plants, its regulation remains largely unknown, when compared with its counterpart in animal cells. In mammalian cells, several pro-apoptotic proteases called caspases were found to play a crucial role in ER stress-induced PCD. Over the past decade, several key proteases with caspase-like enzymatic activity have been discovered in plants and implicated in PCD regulation. This review covers what is known about caspase-like enzymatic activities during plant ER stress-induced PCD and discusses possible regulation pathways leading to the activation of relevant proteases in plants.
PMCID: PMC3924713  PMID: 24592269
programmed cell death; VPE; proteasome; UPR; plant caspases
13.  An Evil Backstage Manipulator: Psychological Factors Correlated with Health-Related Quality of Life in Chinese Patients with Crohn's Disease 
The Scientific World Journal  2013;2013:464698.
Health-related quality of life (HRQoL) is recommended as one of essential parameters to evaluate treatment effect and clinical outcome in patients with Crohn's disease (CD). Recent studies reported that psychological factors might play a role in HRQoL in Western and American CD patients. Sufficient evidences in Chinese CD patients are still unavailable. This study is dedicated to investigate the correlation of various psychological factors with HRQoL in Chinese CD patients. We prospectively collected 40 active and 40 quiescent CD patients in China and found that psychological factors, especially neuroticism and anxiety, significantly correlate with and affect HRQoL in both active and quiescent CD groups. This is the first report revealing correlation between psychological factors and HRQoL in Chinese CD patients. Therefore, we assume that our results can contribute to a better understanding of etiology and tailoring of management in Chinese patients with Crohn's disease and are beneficial to our colleagues to compare the heterogeneous characteristics of Crohn's disease in different ethnic groups.
PMCID: PMC3888728  PMID: 24453858
14.  Crystallization and preliminary X-ray structure analysis of human ribosomal protein L30e 
The purification, crystallization and X-ray structure analysis of human L30e are presented here.
Many functions have been reported for the eukaryotic ribosomal protein L30e. L30e makes several inter-subunit and intra-subunit interactions with protein or RNA components of the 80S ribosome. Yeast L30e has been shown to bind to its own transcript to autoregulate expression at both the transcriptional and the translational levels. Furthermore, it has been reported that mammalian L30e is a component of the selenocysteine-incorporation machinery by binding to the selenocysteine-insertion sequence on mRNA. As high-resolution crystal structures of mammalian L30e are not available, the purification, crystallization and X-ray structure analysis of human L30e are presented here.
PMCID: PMC3232128  PMID: 22139155
L30e; ribosomal proteins
15.  Inhibition of Annexin A2 gene transcription is a promising molecular target for hepatoma cell proliferation and metastasis 
Oncology Letters  2013;7(1):28-34.
Hepatocyte Annexin A2 (ANXA2) expression is associated with the progression and metastasis of hepatocellular carcinoma (HCC). Circulating ANXA2 levels in HCC patients are significantly higher compared with that of patients with benign liver disease. ANXA2 levels have been found to correlate with hepatitis B virus infection, extrahepatic metastasis and portal vein thrombus. By contrast, ANXA2 levels do not correlate with tumour size and AFP levels. However, the underlying mechanisms of ANXA2 remain obscure. The results of the current study identified that abnormalities in hepatic ANXA2 expression were localised to the cell membrane and cytoplasm of HCC tissues and mainly in the cytoplasm of para-cancerous tissues. ANXA2 was overexpressed in MHCC97-H cells which have high metastatic potential. Following specific ANXA2-small hairpin RNA (shRNA) transfection in vitro, ANXA-2 was effectively inhibited and the S phase ratio of cells was 27.76%, compared with 36.14% in mock-treated cells. In addition, the invading cell ratio was reduced in the shRNA-treated group (52.16%) compared with the mock-treated group (86.14%). The growth and volume of xenograft tumours in vivo was significantly suppressed (P<0.05) in the shRNA group compared with that of the mock group, indicating that ANXA2 may be a novel and useful target for elucidating molecular mechanisms involving the proliferation and metastasis of HCC.
PMCID: PMC3861549  PMID: 24348815
hepatocellular carcinoma; Annexin A2; upregulation; small hairpin RNA; hepatitis B virus; metastasis
16.  Differentiation of human amniotic epithelial cells into corneal epithelial-like cells in vitro 
To explore the feasibility that human amniotic epithelial cells (hAECs) have the potential to differentiate into corneal epithelial-like cells under the microenvironment replicated by spontaneously immortalized human corneal epithelial cells (S-ihCECs).
hAECs were isolated by enzyme digestion, and flow cytometry was used to analysis the expression of CD29/90/166/73/34 and HLA-DR. Recovered and cultured S-ihCECs, immunocytochemistry was used to detect the expression of CK3/12. The proliferation of S-ihCECs handled by different concentrations of mitomycin was detected by CCK-8. The proliferation of hAECs cultured by S-ihCECs culture media collected at different time was analyzed by CCK-8. After filtered out the optimal conditions, we collected S-ihCECs culture media for 5 days, then prepared conditioned medium to incubate hAECs, inverted phase contrast microscope and scanning electron microscope were used to observe the change of morphology in hAECs. Quantitative real-time reverse transcription-polymerase chain reaction (QRT-PCR) was carried out to evaluate the expression of Oct-4, NANOG, PAX6, and CK12 in the differentiation period. Immunocytochemistry and western bloting were used to detect the expression of CK3/12.
The culture media collected every 12h, from 20µg/mL mitomycin pretreatment S-ihCECs could significantly promote the proliferation of hAECs. In the period of differentiation, the morphology of differentiated hAECs was obviously different compared with the control group, and the distinctive CK3/12 for corneal epithelial cells was detected.
This study showed that hAECs can differentiate into corneal epithelial-like cells by in vitro replication of the corneal epithelial microenvironment, using the culture media collected from S-ihCECs, and it is possible that S-ihCECs culture media could be used in corneal tissue engineering.
PMCID: PMC3808898  PMID: 24195026
human amniotic epithelial cells; spontaneously immortalized human corneal epithelial cells; mytomicin; microenvironment; tissue engineering
17.  Crystal structure analysis, overexpression and refolding behaviour of a DING protein with single mutation 
Journal of Synchrotron Radiation  2013;20(Pt 6):854-858.
Crystals of a member of the DING protein family (HPBP) were obtained accidentally, and the structure was determined at 1.35 Å resolution. For further analysis, a system for preparation of HPBP was constructed and the structure of a prepared sample was confirmed by X-ray crystal structure analysis at 1.03 Å resolution.
After crystallization of a certain protein–RNA complex, well diffracting crystals were obtained. However, the asymmetric unit of the crystal was too small to locate any components. Mass spectrometry and X-ray crystal structure analysis showed that it was a member of the DING protein family (HPBP). Surprisingly, the structure of HPBP reported previously was also determined accidentally as a contaminant, suggesting that HPBP has a strong tendency to crystallize. Furthermore, DING proteins were reported to relate in disease. These observations suggest that DING has potential for application in a wide range of research fields. To enable further analyses, a system for preparation of HPBP was constructed. As HPBP was expressed in insoluble form in Escherichia coli, it was unfolded chemically and refolded. Finally, a very high yield preparation method was constructed, in which 43 mg of HPBP was obtained from 1 L of culture. Furthermore, to evaluate the validity of refolding, its crystal structure was determined at 1.03 Å resolution. The determined structure was identical to the native structure, in which two disulfide bonds were recovered correctly and a phosphate ion was captured. Based on these results, it was concluded that the refolded HPBP recovers its structure correctly.
PMCID: PMC3795543  PMID: 24121327
DING; HPBP; crystal structure; overexpression; refolding
18.  Overexpression of insulin-like growth factor-I receptor as a pertinent biomarker for hepatocytes malignant transformation 
AIM: To investigate the dynamic features of insulin-like growth factor-I receptor (IGF-IR) expression in rat hepatocarcinogenesis, and the relationship between IGF-IR and hepatocytes malignant transformation at mRNA or protein level.
METHODS: Hepatoma models were made by inducing with 2-fluorenylacetamide (2-FAA) on male Sprague-Dawley rats. Morphological changes of hepatocytes were observed by pathological Hematoxylin and eosin staining, the dynamic expressions of liver and serum IGF-IR were quantitatively analyzed by an enzyme-linked immunosorbent assay. The distribution of hepatic IGF-IR was located by immunohistochemistry. The fragments of IGF-IR gene were amplified by reverse transcription-polymerase chain reaction, and confirmed by sequencing.
RESULTS: Rat hepatocytes after induced by 2-FAA were changed dynamically from granule-like degeneration, precancerous to hepatoma formation with the progressing increasing of hepatic mRNA or IGF-IR expression. The incidences of liver IGF-IR, IGF-IR mRNA, specific IGF-IR concentration (ng/mg wet liver), and serum IGF-IR level (ng/mL) were 0.0%, 0.0%, 0.63 ± 0.17, and 1.33 ± 0.47 in the control; 50.0%, 61.1%, 0.65 ± 0.2, and 1.51 ± 0.46 in the degeneration; 88.9%, 100%, 0.66 ± 0.14, and 1.92 ± 0.29 in the precancerosis; and 100%, 100%, 0.96 ± 0.09, and 2.43 ± 0.57 in the cancerous group, respectively. IGF-IR expression in the cancerous group was significantly higher (P < 0.01) than that in any of other groups at mRNA or protein level. The closely positive IGF-IR relationship was found between livers and sera (r = 0.91, t = 14.222, P < 0.01), respectively.
CONCLUSION: IGF-IR expression may participate in rat hepatocarcinogenesis and its abnormality should be an early marker for hepatocytes malignant transformation.
PMCID: PMC3785631  PMID: 24106410
Hepatoma; Insulin-like growth factor-I receptor; Immunohistochemistry; Gene amplification; Sequencing; Rat hepatoma model
19.  Circulating specific biomarkers in diagnosis of hepatocellular carcinoma and its metastasis monitoring 
Tumour Biology  2013;35(1):9-20.
Hepatocellular carcinoma (HCC) is one of the most common and rapidly fatal malignancies worldwide with a multifactorial, multistep, complex process and poor prognosis. Its early diagnosis and metastasis monitoring are of the utmost importance. Hepatoma tissues synthesize various tumor-related proteins, genes, enzymes, microRNA, etc. and then secrete into the blood. Detections of circulating biomarkers are useful to find tumor at an early stage or monitor metastasis after postoperative treatment. This paper summarizes recent studies of specific biomarkers at early diagnosis or in monitoring metastasis or postoperative recurrence of HCC.
PMCID: PMC3907675  PMID: 24006223
Hepatocellular carcinoma; Specific biomarkers; Diagnosis; Monitoring metastasis; Postoperative recurrence
20.  Associations of Mitochondrial Haplogroups B4 and E with Biliary Atresia and Differential Susceptibility to Hydrophobic Bile Acid 
PLoS Genetics  2013;9(8):e1003696.
Mitochondrial dysfunction has been implicated in the pathogenesis of biliary atresia (BA). This study aimed to determine whether a specific mitochondrial DNA haplogroup is implicated in the pathogenesis and prognosis of BA. We determined 40 mitochondrial single nucleotide polymorphisms in 15 major mitochondrial haplogroups by the use of 24-plex PCR and fluorescent beads combined with sequence-specific oligonucleotide probes in 71 patients with BA and in 200 controls in the Taiwanese population of ethnic Chinese background. The haplogroup B4 and E prevalence were significantly lower and higher respectively, in the patients with BA than in the controls (odds ratios, 0.82 [p = 0.007] and 7.36 [p = 0.032] respectively) in multivariate logistic-regression analysis. The 3-year survival rate with native liver was significantly lower in haplogroup E than the other haplogroups (P = 0.037). A cytoplasmic hybrid (cybrid) was obtained from human 143B osteosarcoma cells devoid of mtDNA (ρ0 cell) and was fused with specific mtDNA bearing E and B4 haplogroups donated by healthy Taiwanese subjects. Chenodeoxycholic acid treatment resulted in significantly lower free radical production, higher mitochondrial membrane potential, more viable cells, and fewer apoptotic cybrid B4 cells than parental 143B and cybrid E cells. Bile acid treatment resulted in a significantly greater protective mitochondrial reaction with significantly higher mitochondrial DNA copy number and mitofusin 1 and 2 concentrations in cybrid B4 and parental cells than in cybrid E cells. The results of the study suggested that the specific mitochondrial DNA haplogroups B4 and E were not only associated with lower and higher prevalence of BA respectively, in the study population, but also with differential susceptibility to hydrophobic bile acid in the cybrid harboring different haplogroups.
Author Summary
Mitochondrial dysfunction has been implicated in the pathogenesis of biliary atresia (BA). We determined 40 mitochondrial single nucleotide polymorphisms in different mitochondrial haplogroups in BA patients and controls. The prevalence of haplogroup B4 and E was significantly lower and higher respectively, in the patients with BA than in the controls. The survival rate with native liver was significantly lower in haplogroup E than the other haplogroups. The in vitro study using cybrid cells revealed significantly lower free radical production, higher mitochondrial membrane potential, higher mitochondrial DNA copy number and fewer apoptotic in cybrid B4 cells than cybrid E cells. The study provides a novel insight into the etiopathogenesis and the predictive value of mitochondrial haplogroups in BA.
PMCID: PMC3744426  PMID: 23966875
21.  Annexin A2 silencing inhibits invasion, migration, and tumorigenic potential of hepatoma cells 
AIM: To investigate the effects of Annexin A2 (ANXA2) silencing on invasion, migration, and tumorigenic potential of hepatoma cells.
METHODS: Human hepatoma cell lines [HepG2, SMMC-7721, SMMC-7402, and MHCC97-H, a novel human hepatocellular carcinoma (HCC) cell line with high metastasis potential] and a normal hepatocyte cell line (LO2) were used in this study. The protein and mRNA expression levels of ANXA2 were analysed by western blotting and real-time polymerase chain reaction, respectively. The intracellular distribution profile of ANXA2 expression was determined by immunofluorescence and immunohistochemistry. Short hairpin RNA targeting ANXA2 was designed and stably transfected into MHCC97-H cells. Cells were cultured for in vitro analyses or subcutaneously injected as xenografts in mice for in vivo analyses. Effects of ANXA2 silencing on cell growth were assessed by cell counting kit-8 (CCK-8) assay (in vitro) and tumour-growth assay (in vivo), on cell cycling was assessed by flow cytometry and propidium iodide staining (in vitro), and on invasion and migration potential were assessed by transwell assay and wound-healing assay, respectively (both in vitro).
RESULTS: The MHCC97-H cells, which are known to have high metastasis potential, showed the highest level of ANXA2 expression among the four HCC cell types examined; compared to the LO2 cells, the MHCC97-H expression level was 8-times higher. The ANXA2 expression was effectively inhibited (about 80%) by ANXA2-specific small hairpin RNA (shRNA). ANXA2 expression in the MHCC97-H cells was mainly localized to the cellular membrane and cytoplasm, and some localization was detected in the nucleus. Moreover, the proliferation of MHCC97-H cells was obviously suppressed by shRNA-mediated ANXA2 silencing in vitro, and the tumour growth inhibition rate was 38.24% in vivo. The percentage of MHCC97-H cells in S phase dramatically decreased (to 27.76%) under ANXA2-silenced conditions. Furthermore, ANXA2-silenced MHCC97-H cells showed lower invasiveness (percentage of invading cells decreased to 52.16%) and suppressed migratory capacity (migration distance decreased to 63.49%). It is also worth noting that shRNA-mediated silencing of ANXA2 in the MHCC97-H cells led to abnormal apoptosis.
CONCLUSION: shRNA-mediated silencing of ANXA2 suppresses the invasion, migration, and tumorigenic potential of hepatoma cells, and may represent a useful target of future molecular therapies.
PMCID: PMC3699036  PMID: 23840117
Annexin A2; Small hairpin RNA; Hepatocellular carcinoma; Invasion; Migration; Tumorigenic potential
22.  Crystallization and preliminary X-ray crystallographic analysis of dihydrouridine synthase from Thermus thermophilus and its complex with tRNA 
Crystals of dihydrouridine synthase from Thermus thermophilus and its complex with tRNA were obtained and X-ray diffraction data were collected to 1.70 and 3.51 Å resolution, respectively.
Dihydrouridine synthase (Dus) is responsible for catalyzing dihydrouridine formation in RNA by the reduction of uridine. To elucidate its RNA-recognition mechanism, Dus from Thermus thermophilus (TthDus) and its complex with tRNA were crystallized. Diffraction data sets were collected from crystals of native and selenomethionine-substituted TthDus to resolutions of 1.70 and 2.30 Å, respectively. These crystals belonged to space group P1. Preliminary X-­ray crystallographic analysis showed that two molecules of TthDus were contained in an asymmetric unit. In addition, diffraction data were collected to 3.51 Å resolution from a crystal of selenomethionine-substituted TthDus in complex with tRNA, which belonged to space group P41212. Preliminary structural analysis showed that the asymmetric unit contained two TthDus–tRNA complexes.
PMCID: PMC3107143  PMID: 21636912
dihydrouridine synthase; tRNA; flavin mononucleotide; Thermus thermophilus
23.  Crystallization and preliminary X-ray crystallographic analysis of eIF5BΔN and the eIF5BΔN–eIF1AΔN complex 
The eukaryotic translation initiation factor eIF5BΔN and the eIF5BΔN–eIF1AΔN complex from S. cerevisiae were crystallized. The crystals diffracted to maximum resolutions of 2.45 and 3.3 Å, respectively.
The binding between two universally conserved translation initiation factors, eIF5B and eIF1A, is important in the initiation step of eukaryotic protein synthesis on the ribosome. Through this interaction, eIF1A assists in recruiting eIF5B to the initiating 40S subunit; eIF5B then encourages the joining of the 60S subunit to form an initiating 80S ribosome. Here, the expression, purification, crystallization and preliminary X-ray analyses of eIF5BΔN and the eIF5BΔN–eIF1AΔN complex from Saccharomyces cerevisiae are reported. The crystal of eIF5BΔN diffracted to 2.45 Å resolution and belonged to space group P41212, with unit-cell parameters a = b = 130.0, c = 71.7 Å. The asymmetric unit was estimated to contain one molecule. The initial phase was obtained by Se-SAD. The crystal of the eIF5BΔN–eIF1AΔN complex diffracted to 3.3 Å resolution and belonged to space group P212121, with unit-cell parameters a = 101.9, b = 120.9, c = 132.8 Å. The asymmetric unit was estimated to contain two complex molecules.
PMCID: PMC3107155  PMID: 21636924
eukaryotic translation initiation; ribosomal subunit joining; initiation factor eIF5B; eIF5B–eIF1A interaction
24.  Crystallization and preliminary X-ray crystallographic study of a methyltransferase involved in 2-methylisoborneol biosynthesis in Streptomyces lasaliensis  
Crystals of a geranyl pyrophosphate methyltransferase in the biosynthetic pathway of the off-flavor terpenoid alcohol, 2-methylisoborneol were obtained in the absence and presence of cofactor, cofactor analog and substrate.
The biosynthetic pathway of the off-flavour terpenoid alcohol 2-methyliso­borneol (2-MIB) requires geranyl pyrophosphate methyltransferase (GPPMT) to methylate GPP before the cyclization reaction. GPPMT is the first example of an S-adenosyl-l-methionine-dependent methyltransferase that acts on general intermediates such as geranyl pyrophosphate and farnesyl pyrophosphate in isoprenoid biosynthetic pathways. In this study, recombinant GPPMT was overproduced, purified and crystallized in the absence and presence of cofactor, cofactor analogue and substrate. Well diffracting crystals of apo GPPMT containing one molecule in the asymmetric unit were obtained and the structure of this form was solved by the molecular-replacement method. Two crystal forms of the tertiary complex with GPP and sinefungin were also obtained. Structure analysis of these crystals is currently under way in order to understand the enzyme reaction mechanism.
PMCID: PMC3053176  PMID: 21393856
geranyl pyrophosphate methyltransferase; 2-methylisoborneol biosynthesis; Streptomyces lasaliensis
25.  Antimicrobial Photodynamic Therapy for Methicillin-Resistant Staphylococcus aureus Infection 
BioMed Research International  2013;2013:159157.
Nowadays methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common multidrug resistant bacteria both in hospitals and in the community. In the last two decades, there has been growing concern about the increasing resistance to MRSA of the most potent antibiotic glycopeptides. MRSA infection poses a serious problem for physicians and their patients. Photosensitizer-mediated antimicrobial photodynamic therapy (PDT) appears to be a promising and innovative approach for treating multidrug resistant infection. In spite of encouraging reports of the use of antimicrobial PDT to inactivate MRSA in large in vitro studies, there are only few in vivo studies. Therefore, applying PDT in the clinic for MRSA infection is still a long way off.
PMCID: PMC3600246  PMID: 23555074

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