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1.  Identification of Valid Reference Genes for the Normalization of RT-qPCR Expression Studies in Human Breast Cancer Cell Lines Treated with and without Transient Transfection 
PLoS ONE  2015;10(1):e0117058.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a powerful technique for examining gene expression changes during tumorigenesis. Target gene expression is generally normalized by a stably expressed endogenous reference gene; however, reference gene expression may differ among tissues under various circumstances. Because no valid reference genes have been documented for human breast cancer cell lines containing different cancer subtypes treated with transient transfection, we identified appropriate and reliable reference genes from thirteen candidates in a panel of 10 normal and cancerous human breast cell lines under experimental conditions with/without transfection treatments with two transfection reagents. Reference gene expression stability was calculated using four algorithms (geNorm, NormFinder, BestKeeper and comparative delta Ct), and the recommended comprehensive ranking was provided using geometric means of the ranking values using the RefFinder tool. GeNorm analysis revealed that two reference genes should be sufficient for all cases in this study. A stability analysis suggests that 18S rRNA-ACTB is the best reference gene combination across all cell lines; ACTB-GAPDH is best for basal breast cancer cell lines; and HSPCB-ACTB is best for ER+ breast cancer cells. After transfection, the stability ranking of the reference gene fluctuated, especially with Lipofectamine 2000 transfection reagent in two subtypes of basal and ER+ breast cell lines. Comparisons of relative target gene (HER2) expression revealed different expressional patterns depending on the reference genes used for normalization. We suggest that identifying the most stable and suitable reference genes is critical for studying specific cell lines under certain circumstances.
doi:10.1371/journal.pone.0117058
PMCID: PMC4305315  PMID: 25617865
2.  TLR3 Signaling in Macrophages Is Indispensable for the Protective Immunity of Invariant Natural Killer T Cells against Enterovirus 71 Infection 
PLoS Pathogens  2015;11(1):e1004613.
Enterovirus 71 (EV71) is the most virulent pathogen among enteroviruses that cause hand, foot and mouth disease in children but rarely in adults. The mechanisms that determine the age-dependent susceptibility remain largely unclear. Here, we found that the paucity of invariant natural killer T (iNKT) cells together with immaturity of the immune system was related to the susceptibility of neonatal mice to EV71 infection. iNKT cells were crucial antiviral effector cells to protect young mice from EV71 infection before their adaptive immune systems were fully mature. EV71 infection led to activation of iNKT cells depending on signaling through TLR3 but not other TLRs. Surprisingly, iNKT cell activation during EV71 infection required TLR3 signaling in macrophages, but not in dendritic cells (DCs). Mechanistically, interleukin (IL)-12 and endogenous CD1d-restricted antigens were both required for full activation of iNKT cells. Furthermore, CD1d-deficiency led to dramatically increased viral loads in central nervous system and more severe disease in EV71-infected mice. Altogether, our results suggest that iNKT cells may be involved in controlling EV71 infection in children when their adaptive immune systems are not fully developed, and also imply that iNKT cells might be an intervention target for treating EV71-infected patients.
Author Summary
Enterovirus 71 (EV71) is a major causative pathogen of hand, foot and mouth disease. EV71 infection occurs mainly in children but rarely in adults. The factors that determine the susceptibility of children to EV71 infection remain elusive. Here, we found that the paucity of invariant natural killer T (iNKT) cells in new-born mice was associated with their susceptibility to EV71 infection. Furthermore, iNKT cells played a critical role in protecting older young mice from EV71 infection before their adaptive immune systems were fully developed. Mechanistically, TLR3 signaling in macrophages, but not in dendritic cells, was essentially required for iNKT cell activation during EV71 infection. Both interleukin (IL)-12 production and endogenous lipid antigens presented by macrophages were required for full iNKT cell activation. iNKT cells tended to prevent the dissemination of EV71 into central nervous system. Taken together, our findings provide a new insight into the susceptibility of children to EV71 infection, and imply that the manipulation of iNKT cells might represent a potential therapeutic strategy for HFMD and other viral infectious diseases in children.
doi:10.1371/journal.ppat.1004613
PMCID: PMC4304831  PMID: 25615690
3.  Novel recombinant chimeric virus-like particle is immunogenic and protective against both enterovirus 71 and coxsackievirus A16 in mice 
Scientific Reports  2015;5:7878.
Hand-foot-and-mouth disease (HFMD) has been recognized as an important global public health issue, which is predominantly caused by enterovirus 71 (EV-A71) and coxsackievirus A16 (CVA16). There is no available vaccine against HFMD. An ideal HFMD vaccine should be bivalent against both EV-A71 and CVA16. Here, a novel strategy to produce bivalent HFMD vaccine based on chimeric EV-A71 virus-like particles (ChiEV-A71 VLPs) was proposed and illustrated. The neutralizing epitope SP70 within the capsid protein VP1 of EV-A71 was replaced with that of CVA16 in ChiEV-A71 VLPs. Structural modeling revealed that the replaced CVA16-SP70 epitope is well exposed on the surface of ChiEV-A71 VLPs. These VLPs produced in Saccharomyces cerevisiae exhibited similarity in both protein composition and morphology as naive EV-A71 VLPs. Immunization with ChiEV-A71 VLPs in mice elicited robust Th1/Th2 dependent immune responses against EV-A71 and CVA16. Furthermore, passive immunization with anti-ChiEV-A71 VLPs sera conferred full protection against lethal challenge of both EV-A71 and CVA16 infection in neonatal mice. These results suggested that this chimeric vaccine, ChiEV-A71 might have the potential to be further developed as a bivalent HFMD vaccine in the near future. Such chimeric enterovirus VLPs provide an alternative platform for bivalent HFMD vaccine development.
doi:10.1038/srep07878
PMCID: PMC4297979  PMID: 25597595
4.  Nonparametric Comparison for Multivariate Panel Count Data 
Multivariate panel count data often occur when there exist several related recurrent events or response variables defined by occurrences of related events. For univariate panel count data, several nonparametric treatment comparison procedures have been developed. However, it does not seem to exist a nonparametric procedure for multivariate cases. Based on differences between estimated mean functions, this paper proposes a class of nonparametric test procedures for multivariate panel count data. The asymptotic distribution of the new test statistics is established and a simulation study is conducted. Also the new procedures are applied to a skin cancer problem that motivated this study.
doi:10.1080/03610926.2012.667486
PMCID: PMC3898619  PMID: 24465081
Counting processes; Medical follow-up study; Nonparametric comparison; Panel count data; Skin cancer study
5.  Heme oxygenase-1 in pregnancy and cancer: similarities in cellular invasion, cytoprotection, angiogenesis, and immunomodulation 
Pregnancy can be defined as a “permissible” process, where a semi-allogeneic fetus and placenta are allowed to grow and survive within the mother. Similarly, in tumor growth, antigen-specific malignant cells proliferate and evade into normal tissues of the host. The microenvironments of the placenta and tumors are amazingly comparable, sharing similar mechanisms exploited by fetal or cancer cells with regard to surviving in a hypoxic microenvironment, invading tissues via degradation and vasculogenesis, and escaping host attack through immune privilege. Heme oxygease-1 (HO-1) is a stress-response protein that has antioxidative, anti-apoptotic, pro-angiogenic, and anti-inflammatory properties. Although a large volume of research has been published in recent years investigating the possible role(s) of HO-1 in pregnancy and in cancer development, the molecular mechanisms that regulate these “yin-yang” processes have still not been fully elucidated. Here, we summarize and compare pregnancy and cancer development, focusing primarily on the function of HO-1 in cellular invasion, cytoprotection, angiogenesis, and immunomodulation. Due to the similarities of both processes, a thorough understanding of the molecular mechanisms of each process may reveal and guide the development of new approaches to prevent not only pregnancy disorders; but also, to study cancer.
doi:10.3389/fphar.2014.00295
PMCID: PMC4294126  PMID: 25642189
Placenta; trophoblast invasion; angiogenesis;  immunosuppression; tolerogenic dendritic cells (tDC); alternatively activated macrophage (M2)
6.  Pigment epithelium-derived factor protects the morphological structure of retinal Müller cells in diabetic rats 
AIM
To investigate if pigment epithelium-derived factor (PEDF) has any protective effect on the retinal Müller cells of Sprague-Dawley rats suffering from diabetes mellitus.
METHODS
Sixty Sprague-Dawley rats were randomly divided into a negative control group, a group receiving 0.1 µg/µL PEDF, another group receiving 0.2 µg/µL PEDF, and a group receiving balanced salt solution (BSS). Rats in both the PEDF and BSS groups were treated intravitreally based on previously established diabetic models. After 4wk of treatment, morphological alterations of Müller cells and protein expression of glutamine synthase (GS) and glial fibrillary acidic protein (GFAP) were analyzed.
RESULTS
PEDF at either 0.1 µg/µL or 0.2 µg/µL significantly improved the structures of both nuclei and organelles of Müller cells compared to the BSS-treated group. Expression of GS was significantly higher in the 0.2 µg/µL PEDF group than that in the BSS group (P=0.012), but expression of GFAP was significantly lower in the 0.2 µg/µL PEDF group than that in the BSS group (P=0.000); however, there were no significant differences in expression of these proteins between the 0.1 µg/µL PEDF group and the BSS group (P=0.608, P=0.152).
CONCLUSION
PEDF protects the morphological ultrastructure of Müller cells, improves the expression of glutamate synthase and prevents cell gliosis.
doi:10.3980/j.issn.2222-3959.2014.06.05
PMCID: PMC4270985  PMID: 25540743
diabetes mellitus; pigment epithelium-derived factor; retinal Müller cells; glutamine synthase; glial fibrillary acidic protein
7.  Pirarubicin-based chemotherapy displayed better clinical outcomes and lower toxicity than did doxorubicin-based chemotherapy in the treatment of non-metastatic extremity osteosarcoma 
Pirarubicin (THP) is a newer generation anthracycline anticancer drug with antineoplastic efficacy against numerous tumors. Few studies have reported its application and efficiency in anti-osteosarcoma chemotherapeutic strategies. Ninety-six non-metastatic extremity osteosarcoma patients treated with THP or doxorubicin (DOX) in combination with high-dose methotrexate (HDMTX), cisplatin (DDP) and ifosfamide (IFO) within the past 9 years at our hospital were evaluated retrospectively to compare efficacy and side effects. Among the patients, 55.2% were male, 36.5% were ≤14 years old and 59.4% presented with a large tumor (≥1/3 of bone) to our department. The 5-year disease-free survival (DFS) rate of the patients treated with the THP-based chemotherapeutic regimen was 70.2%, significantly higher than that of the DOX-based regimen-treated group (53.1%). The THP-based chemotherapeutic regimen decreased the lung metastatic rate significantly compared with the DOX-based regimen (19.1% vs. 36.7%, P=0.045), as well as the relapse rate (31.9% vs. 49.0%, P=0.067). Both regimens were generally well tolerated. However, while the THP-based chemotherapeutic regimen did not alter toxicity in the hematologic system, liver or kidneys compared with the DOX-based regimen, it showed lower rates of alopecia (63.8% vs. 85.7%, P=0.012), nausea and vomiting (51.1% vs. 79.6%, P=0.003), and mucositis (48.9% vs. 75.6%, P=0.003). THP also resulted in lower cardiac toxicity. Our data demonstrate that the THP-based regimen is better than the DOX-based regimen in terms of the 5-year DFS rate, pulmonary metastasis rate, relapse rate and side effects.
PMCID: PMC4300710  PMID: 25628949
Osteosarcoma; chemotherapy; pirarubicin; doxorubicin; relapse; side effects; disease-free survival; overall survival
8.  A retrospective study of risk and prognostic factors in relation to lower respiratory tract infection in elderly lung cancer patients 
Lung cancer (LCa) is one of the most common and deadly malignancies in elderly patients. During the course of the disease, these patients frequently present with lower respiratory tract infection. Therefore, this study aims to investigate the clinical features of lower respiratory tract infection in elderly LCa patients and evaluate the impact on overall survival rate. Clinical and laboratory data were analyzed retrospectively for a total of 1936 patients that were over 60-years-old. Patients were classified into three groups based on pulmonary diseases: Group 1, lung cancer (LCa); Group 2, chronic obstructive pulmonary disease (COPD); and Group 3, other medical diseases without pulmonary problems (OMD). Univariate and multivariate analysis were used to evaluate related risk factors of infections and prognostic factors. The infection rate of the LCa group (46.25%) was significantly higher than the COPD (31.40%) and OMD (23.33%) groups. Polymicrobial infections were most prevalent in the LCa group (28.75%), which far exceeded the prevalence in COPD (11.05%) and OMD (4.44%) groups. In LCa patients, the most frequent pathogens were Gram-negative bacteria (44.87%), followed by fungi (34.62%) and Gram-positive bacteria (20.51%), the major pattern of polymicrobial infections was mixed Gram-negative bacteria and fungi (43.48%). Multivariate analysis revealed that COPD, pleural effusion, anatomical type, low cellular immune function, and length of hospital stay were related risk factors of lower respiratory tract infection in elderly LCa patients. A multivariate Cox proportional hazards regression model revealed that age, stage of TNM, surgical resection, antitumor therapy, lower respiratory tract infection, COPD, and pleural effusion were independent prognostic factors for cancer-related death. Patients who received effective antimicrobial treatment had a better outcome than those who did not respond to antimicrobial drugs (HR = 0.458, P < 0.05). Understanding lower respiratory tract infection in elderly LCa patients is vital if we are to set up corresponding measures and to target effective antimicrobial treatment.
PMCID: PMC4300720  PMID: 25628950
Lung cancer; infection; risk factors; prognosis
9.  Adenovirus-delivered PDCD5 counteracts adriamycin resistance of osteosarcoma cells through enhancing apoptosis and inhibiting Pgp 
In the present study, we investigated the roles of PDCD5 (programmed cell death 5) in multidrug re-sistance (MDR) of osteosarcoma cells and the possible lurking mechanisms. An adenovirus expression vector of PDCD5 was constructed and transfected into human adriamycin-resistant osteosarcoma cell line Saos-2/ADM. We found that up-regulation of PDCD5 could significantly enhance the sensitivity of Saos-2/ADM cells towards vincristine, methotrexate, cisplatin and arsenic trioxide (As2O3), and could decrease the capacity of cells to efflux adriamycin. PDCD5 could significantly down regulate the expression of P-glycoprotein (Pgp), but not affect the expression of multidrug resistance associated protein (MRP) or the glutathione S-transferase (GST). PDCD5 was also able to significantly increase the apoptotic activity of modified osteosarcoma cells. Further study of the biological functions of PDCD5 might be helpful in the understanding of the mechanisms of multidrug resistance (MDR) in osteosarcoma and exploring PDCD5 based adjuvant genetic therapy.
PMCID: PMC4307499  PMID: 25664052
Osteosarcoma; MDR; PDCD5; apoptosis
10.  Effects of Maternal LPS Exposure during Pregnancy on Metabolic Phenotypes in Female Offspring 
PLoS ONE  2014;9(12):e114780.
It is increasingly recognized that intra-uterine growth restriction (IUGR) is associated with an increased risk of metabolic disorders in late life. Previous studies showed that mice exposed to LPS in late gestation induced fetal IUGR. The present study investigated the effects of maternal LPS exposure during pregnancy on metabolic phenotypes in female adult offspring. Pregnant mice were intraperitoneally injected with LPS (50 µg/kg) daily from gestational day (GD)15 to GD17. After lactation, female pups were fed with standard-chow diets (SD) or high-fat diets (HFD). Glucose tolerance test (GTT) and insulin tolerance test (ITT) were assessed 8 and 12 weeks after diet intervention. Hepatic triglyceride content was examined 12 weeks after diet intervention. As expected, maternal LPS exposure during pregnancy resulted in fetal IUGR. Although there was an increasing trend on fat mass in female offspring whose dams were exposed to LPS during pregnancy, maternal LPS exposure during pregnancy did not elevate the levels of fasting blood glucose and serum insulin and hepatic triglyceride content in female adult offspring. Moreover, maternal LPS exposure during pregnancy did not alter insulin sensitivity in adipose tissue and liver in female adult offspring. Further analysis showed that maternal LPS exposure during pregnancy did not exacerbate HFD-induced glucose tolerance and insulin resistance in female adult offspring. In addition, maternal LPS exposure during pregnancy did not aggravate HFD-induced elevation of hepatic triglyceride content in female adult offspring. In conclusion, LPS-induced IUGR does not alter metabolic phenotypes in adulthood.
doi:10.1371/journal.pone.0114780
PMCID: PMC4257726  PMID: 25479255
11.  Zoonotic parapoxviruses detected in symptomatic cattle in Bangladesh 
BMC Research Notes  2014;7(1):816.
Background
Application of molecular diagnostic methods to the determination of etiology in suspected poxvirus-associated infections of bovines is important both for the diagnosis of the individual case and to form a more complete understanding of patterns of strain occurrence and spread. The objective of this study was to identify and characterize bovine-associated zoonotic poxviruses in Bangladesh which are relevant to animal and human health.
Findings
Investigators from the International Center Diarrhoeal Disease Research (icddr,b), the US Centers for Disease Control and Prevention (CDC), and the Bangladesh Department of Livestock Services traveled to three districts in Bangladesh—Siranjganj, Rangpur and Bhola–to collect diagnostic specimens from dairy cattle and buffalo that had symptoms consistent with poxvirus-associated infections. Bovine papular stomatitis virus (BPSV) DNA was obtained from lesion material (teat) and an oral swab collected from an adult cow and calf (respectively) from a dairy production farm in Siranjganj. Pseudocowpox virus (PCPV) DNA signatures were obtained from a scab and oral swab collected from a second dairy cow and her calf from Rangpur.
Conclusions
We report the first detection of zoonotic poxviruses from Bangladesh and show phylogenetic comparisons between the Bangladesh viruses and reference strains based on analyses of the B2L and J6R loci (vaccinia orthologs). Understanding the range and diversity of different species and strains of parapoxvirus will help to spotlight unusual patterns of occurrence that could signal events of significance to the agricultural and public health sectors.
doi:10.1186/1756-0500-7-816
PMCID: PMC4246640  PMID: 25410770
Bangladesh; Bovine papular stomatitis virus; Parapoxvirus; Zoonosis; Pseudocowpox virus
12.  Platelets induce a proinflammatory phenotype in monocytes via the CD147 pathway in rheumatoid arthritis 
Introduction
Activated platelets exert a proinflammatory action that can be largely ascribed to their ability to interact with monocytes. However, the mechanisms that promote dynamic changes in monocyte subsets in rheumatoid arthritis (RA) have not been clearly identified. The aim of this study was to determine whether platelet activation and the consequent formation of monocyte-platelet aggregates (MPA) might induce a proinflammatory phenotype in circulating monocytes in RA.
Methods
The surface phenotype of platelets and the frequencies of monocyte subpopulations in the peripheral blood of RA patients were determined using flow cytometry. Platelets were sorted and co-cultured with monocytes. In addition, monocyte activation was assessed by measuring the nuclear factor kappa B (NF-κB) pathway. The disease activity was evaluated using the 28-joint disease activity score.
Results
Platelet activation, circulating intermediate monocytes (Mon2) and MPA formation were significantly elevated in RA, especially in those with active disease status. Furthermore, Mon2 monocytes showed higher CD147 expression and responded to direct cell contact with activated platelets with higher cytokine production and matrix metallopeptidase 9 (MMP-9) secretion, which increased the expression of CD147. After the addition of specific antibodies for CD147, those effects were abolished. Furthermore, the NF-κB-driven inflammatory pathway may be involved in this process.
Conclusions
These findings indicate an important role of platelet activation and the consequent formation of MPA in the generation of the proinflammatory cytokine milieu and for the promotion and maintenance of the pathogenically relevant Mon2 monocyte compartment in RA, which is likely to play an important role in the pathogenesis of autoimmunity.
doi:10.1186/s13075-014-0478-0
PMCID: PMC4298113  PMID: 25404518
13.  Effects of pamidronate disodium on the loss of osteoarthritic subchondral bone and the expression of cartilaginous and subchondral osteoprotegerin and RANKL in rabbits 
Background
Osteoarthritis (OA) is a major health problem in the increasingly elderly population. Therefore, it is crucial to prevent and treat OA at an early stage. The present study investigated whether pamidronate disodium (PAM), a bone-loss inhibitor, can significantly prevent or reverse the progression of early anterior cruciate ligament transection (ACLT)-induced OA. Whether therapeutic intervention is associated with regulation of the expression of osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand (RANKL), metalloproteinase-9 (MMP-9) or Toll-like receptor-4 (TLR-4) in cartilage and/or subchondral bone was also investigated.
Methods
60 New Zealand rabbits were randomized into four groups: Sham-operated (n = 20); ACLT (n = 20); short-term treatment with PAM (PAM-S, n = 10) and long-term treatment with PAM (PAM-L, n = 10). For cartilage and subchondral bone testing, rabbits from Sham and ACLT groups were harvested at 2, 4, 6, and 14 weeks. Rabbits were given PAM from the 4th week after ACLT operation in PAM-S and PAM-L group, and were harvested at 6 and 14 weeks, respectively. Trabecular characteristics and cartilage changes were detected using Micro-CT, safranin O and rapid green staining, respectively. Immunohistochemical staining for OPG and RANKL were also performed. OPG, RANKL, MMP-9 and TLR-4 expression was evaluated by western blot analysis.
Results
Micro-CT and histology analyses indicated that PAM treatment for 2 or 10 weeks could completely prevent or reverse osteoarthritic subchondral bone loss and cartilage surface erosion. Immunohistochemistry and western blot analysis indicated that expression of OPG and RANKL increased, although RANKL expression increased more significantly than that of OPG. Therefore the ratio of OPG to RANKL was lower in the ACLT group. However, the ratio of OPG to RANKL in the PAM group was significantly higher than that in the ACLT group. Additionally, expression of MMP-9 and TLR-4 were upregulated in the ACLT group and downregulated in the PAM treated groups.
Conclusions
PAM can significantly inhibit and even reverse early osteoarthritic subchondral bone loss, thus alleviating the process of cartilaginous degeneration. The mechanisms involved may be associated with the upregulation of OPG expression, and downregulation of RANKL, MMP-9 and TLR-4 expression.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2474-15-370) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2474-15-370
PMCID: PMC4240862  PMID: 25377946
Osteoarthritis; Pamidronate disodium; Subchondral bone; Cartilage; Osteoprotegerin (OPG); Receptor activator of nuclear factor-κ B ligand (RANKL)
14.  Silencing of insulin-like growth factor-1 receptor enhances the radiation sensitivity of human esophageal squamous cell carcinoma in vitro and in vivo 
Background
Esophageal squamous cell carcinoma (ESCC) is a prevalent fatal cancer worldwide, and the number of deaths due to this disease is increasing. Due to ESCC resistance to chemotherapy and radiation treatment, new therapies are urgently needed for the improvement of ESCC patient clinical outcomes.
Methods
Eca-109 and TE-1 cells were transfected with 100 nM IGF-1r siRNA, and a combination of IGF-1r siRNA and radiation therapy was tested in vitro and in vivo. The effects of IGF-1r siRNA were determined through Western blotting and flow cytometry experiments.
Results
After radiotherapy, the number of IGF-1r siRNA-transfected Eca-109 cells decreased by approximately 67.3%, and a 78.9% reduction was observed in the transfected TE-1 cells. In addition, the Eca-109 and TE-1 cells that were irradiated following IGF-1r knockdown contained 16.2% and 20.3% apoptotic cells, respectively.
Conclusions
The results of the current study suggest that IGF-1r knockdown may enhance the radiation sensitivity of ESCC and increase the therapeutic effects of radiation both in vitro and in vivo. These results provide strong evidence that the targeted application of siRNA will enable the development of new therapeutic strategies for the clinical treatment of ESCC patients.
doi:10.1186/1477-7819-12-325
PMCID: PMC4232704  PMID: 25363593
Human esophageal squamous cell carcinoma; Radiation sensitivity; IGF-1r; RNAi
15.  Crystallization and preliminary crystallographic studies of the W2 domain of Drosophila melanogaster eukaryotic translation initiation factor 5C domain-containing protein 
The crystallization and preliminary crystallographic studies of the carboxy-terminal domain of D. melanogaster eukaryotic translation initiation factor 5C domain-containing protein are reported.
The Drosophila melanogaster eukaryotic translation initiation factor 5C domain-containing protein (ECP) is composed of two independently folded domains which belong to the basic leucine-zipper and W2 domain-containing protein (BZW) family. Based on the sequence similarity between the C-terminal W2 domain of ECP and some eukaryotic translation initiation factors (such as eIF2B∊, eIF4γ, eIF5 etc.), ECP has been speculated to participate in the translation initiation process. Structural information on the C-terminal W2 domain of ECP would be helpful in understanding the specific cellular function of this protein. Here, the W2 domain of ECP was expressed and crystallized. Crystals grown by the hanging-drop vapour-diffusion method diffracted to 2.70 Å resolution and belonged to space group I4, with unit-cell parameters a = b = 81.05, c = 57.44 Å. The Matthews coefficient suggested that there was one molecule per asymmetric unit in the crystal.
doi:10.1107/S1744309112036512
PMCID: PMC3515371  PMID: 23143239
ECP; eIF5C; W2 domain; Drosophila melanogaster
16.  Clinical study of customized aspherical intraocular lens implants 
AIM
To compare if there is an improvement in visual functions with age-related cataracts between patients receiving a aspherical intraocular lens (IOL) based on corneal wavefront aberration and patients randomly assigned lenses.
METHODS
A total of 124 eyes of 124 patients with age-related cataracts were placed in experimental group and a group receiving randomly assigned (RA) lenses. The experimental group was undergone Pentacam corneal spherical aberration measurement before surgery; the targeted range for residual total spherical aberration after surgery was set to 0-0.3 µm. Patients with a corneal spherical aberration <0.3 µm were implanted with a zero-spherical aberration advanced optics (AO) aspherical IOL and patients with an aberration ≥0.3 µm received a Tecnis Z9003 aspherical lens in experimental group. RA patients were randomly implanted with an AO lens or a Tecnis Z9003 lens. Three months after surgery total spherical aberration, photopic/mesopic contrast sensitivities, photopic/mesopic with glare contrast sensitivities, and logMAR vision were measured.
RESULTS
Statistical analysis on logMAR vision showed no significant difference between two groups (P=0.413). The post-surgical total spherical aberration was 0.126±0.097 µm and 0.152±0.151 µm in the experimental and RA groups, respectively (P=0.12). The mesopic contrast sensitivities at spatial frequencies of 6, 12 and 18 c/d in the experimental group were significantly higher than of the RA group (P=0.00; P=0.04; P=0.02). The mesopic with glare contrast sensitivity in the experimental group at a spatial frequency of 18 c/d was also significantly higher vs the RA group (P=0.01).
CONCLUSION
Pre-surgical corneal spherical aberration measurement in cataract patients followed by customized selection of aspherical IOL implants improved mesopic contrast sensitivities at high spatial frequencies, and thus is a superior strategy compared to the random selection of aspherical IOL implants.
doi:10.3980/j.issn.2222-3959.2014.05.14
PMCID: PMC4206887  PMID: 25349799
intraocular lens; cataract extraction; corneal wavefront aberration; mesopic vision; night vision
17.  MicroRNAs in cancer therapeutic response: Friend and foe 
Cancer initiation and development engage extremely complicated pathological processes which involve alterations of a large number of cell signaling cascades and functional networks in temporal and spatial orders. During last decades, microRNAs (miRNAs), a class of non-coding RNAs, have emerged as critical players in cancer pathogenesis and progression by modulating many pathological aspects related to tumor development, growth, metastasis, and drug resistance. The major function of miRNAs is to post-transcriptionally regulate gene expression depending on recognition of complementary sequence residing in target mRNAs. Commonly, a particular miRNA recognition sequence could be found in a number of genes, which allows a single miRNA to regulate multiple functionally connected genes simultaneously and/or chronologically. Furthermore, a single gene can be targeted and regulated by multiple miRNAs. However, previous studies have demonstrated that miRNA functions are highly context-dependent, which leads to distinct pathological outcomes in different types of cancer as well as at different stages by alteration of the same miRNA. Here we summarize recent progress in studies on miRNA function in cancer initiation, metastasis and therapeutic response, focusing on breast cancer. The varying functions of miRNAs and potential application of using miRNAs as biomarkers as well as therapeutic approaches are further discussed in the context of different cancers.
doi:10.5306/wjco.v5.i4.730
PMCID: PMC4129536  PMID: 25302173
MicroRNA; Breast cancer; Therapeutic response; Biomarker
18.  The involvement of high mobility group 1 cytokine and phospholipases A2 in diabetic retinopathy 
Background
Diabetic retinopathy, the main microvascular complications of diabetes and one of the leading causes of blindness worldwide. Interesting reports on the role of inflammatory/proangiogenic high mobility group 1 (HMGB-1) cytokine and phospholipases A2 (PLA2) in neovascularization have diverted our concentration to reveal whether HMGB-1 and PLA2 plays role in diabetic retinopathy.
Methods
We performed our study in streptozotocin (STZ)-induced diabetic rat model. The expression levels of the cytokines, chemokines, and cell adhesion molecules in retinal tissues were evaluated by quantitative RT-PCR. HMGB-1 and PLA2 protein levels along with VEGF, TNF-α, IL-1β and ICAM-1 levels were also measured.
Results
We observed the retinal pericytes, endothelial injury/death and breakdown of blood–retinal barrier (BRB). The protein expression of HMGB-1, PLA2 and IL-1β were significantly increased in micro vessels from retina of diabetic rats. Diabetic rats had also high retinal levels of VEGF, ICAM-1 and TNF-α. Further investigation revealed that pericyte death is mediated by HMGB-1-induced cytotoxic activity of glial cells, while HMGB-1 can directly mediate endothelial cell death. Similarly, increased expression of PLA2 represents the diabetic mediated alteration of BRB, perhaps up regulating the VEGF.
Conclusions
Our data suggest that HMGB-1 and PLA2 involved in retinal pericyte and endothelial injury and cell death in diabetic retinopathy. From this study, we suggest that HMGB-1 and PLA2 may be interesting targets in managing diabetic retinopathy.
doi:10.1186/1476-511X-13-156
PMCID: PMC4287419  PMID: 25292258
Blood retinal barrier; Micro vessels; Retinal pericytes; Endothelial cells
19.  Surgical management of cervical esophageal carcinoma with larynx preservation and reconstruction 
Objectives: There is no generally accepted treatment strategy for cervical esophageal carcinoma. The purpose of this study was to evaluate the operative outcomes of reconstruction after resection of cervical esophageal and hypopharynx-esophagus junction carcinoma with larynx preservation. Methods: We retrospectively reviewed the data of 79 patients with carcinoma of the hypopharynx-esophagus junction and cervical esophagus. Transhiatal total esophagectomy without thoracotomy was carried out in 67 patients who underwent gastric pull-up (GP) or colon interposition (CI) techniques. Transcervical limited pharyngo-cervical esophagectomy was performed in the patients with the pectoralis major flap alone or combined with the split graft (PMF/CWSG) for reconstruction. Seventy-two patients received postoperative adjuvant therapy. Results: The 3-year and 5-year overall survival rates were 66.4% and 45.5%, respectively. The average time to resumption of oral feeding was 25.2 days. All patients had preserved laryngeal function. The overall incidence of complications was 29.1% (23/79), which included cervical fistula, abdominal wound dehiscence, liquefaction necrosis of abdominal fat, and pleural effusion. Conclusions: Surgical resection of cervical esophageal carcinoma and laryngeal preservation is possible. Complete esophagectomy should be performed when the resection extends below the thoracic inlet. The reconstruction methods we performed were safe and effective for the immediate restoration of alimentary continuity after resection of cervical esophageal and pharyngo-cervical esophageal carcinoma; and the patients with PMF/CWSG reconstruction had a better survival than those with GP or CI reconstruction. Combined with radiotherapy, the resectability rate and survival rate of cervical esophageal carcinoma can be improved.
PMCID: PMC4211788  PMID: 25356138
Carcinoma; cervical esophagus; gastric pull-up; colon interposition; larynx preservation; oral cancer
20.  The Importance of Hydrogen Bonding and Aromatic Stacking to the Affinity and Efficacy of Cannabinoid Receptor CB2 Antagonist, 5-(4-Chloro-3-methyl-phenyl)-1-(4-methyl-benzyl)-1H-pyrazole-3-carboxylic acid (1,3,3-trimethyl-bicyclo[2.2.1]hept-2-yl)-amide (SR144528) 
Journal of medicinal chemistry  2013;56(17):6593-6612.
Despite the therapeutic promise of the sub-nanomolar affinity cannabinoid CB2 antagonist, N-[(1S)-endo-1,3,3-trimethylbicyclo[2.2.1]heptan2-yl]-5-(4-chloro-3-methylphenyl)-1-[(4-methylphenyl)methyl]-1H-pyrazole-3-carboxamide (SR144528, 1), little is known about its binding site interactions and no primary interaction site for 1 at CB2 has been identified. We report here the results of Glide docking studies in our cannabinoid CB2 inactive state model that were then tested via compound synthesis, binding and functional assays. Our results show that the amide functional group of 1 is critical to its CB2 affinity and efficacy and that aromatic stacking interactions in the TMH5/6 aromatic cluster of CB2 are also important. Molecular modifications that increased the positive electrostatic potential in the region between the fenchyl and aromatic rings led to more efficacious compounds. This result is consistent with the EC-3 loop negatively charged amino acid, D275 (identified via Glide docking studies) acting as the primary interaction site for 1 and its analogs.
doi:10.1021/jm400070u
PMCID: PMC3804063  PMID: 23855811
21.  Novel HIV-1 MiRNAs Stimulate TNFα Release in Human Macrophages via TLR8 Signaling Pathway 
PLoS ONE  2014;9(9):e106006.
Purpose
To determine whether HIV-1 produces microRNAs and elucidate whether these miRNAs can induce inflammatory response in macrophages (independent of the conventional miRNA function in RNA interference) leading to chronic immune activation.
Methods
Using sensitive quantitative Real Time RT-PCR and sequencing, we detected novel HIV-derived miRNAs in the sera of HIV+ persons, and associated with exosomes. Release of TNFα by macrophages challenged with HIV miRNAs was measured by ELISA.
Results
HIV infection of primary alveolar macrophages produced elevated levels of viral microRNAs vmiR88, vmiR99 and vmiR-TAR in cell extracts and in exosome preparations from conditioned medium. Furthermore, these miRNAs were also detected in exosome fraction of sera from HIV-infected persons. Importantly, vmiR88 and vmiR99 (but not vmiR-TAR) stimulated human macrophage TNFα release, which is dependent on macrophage TLR8 expression. These data support a potential role for HIV-derived vmiRNAs released from infected macrophages as contributing to chronic immune activation in HIV-infected persons, and may represent a novel therapeutic target to limit AIDS pathogenesis.
Conclusion
Novel HIV vmiR88 and vmiR99 are present in the systemic circulation of HIV+ persons and could exhibit biological function (independent of gene silencing) as ligands for TLR8 signaling that promote macrophage TNFα release, and may contribute to chronic immune activation. Targeting novel HIV-derived miRNAs may represent a therapeutic strategy to limit chronic immune activation and AIDS progression.
doi:10.1371/journal.pone.0106006
PMCID: PMC4156304  PMID: 25191859
22.  Tripartite Interactions of Barley Yellow Dwarf Virus, Sitobion avenae and Wheat Varieties 
PLoS ONE  2014;9(9):e106639.
The tripartite interactions in a pathosystem involving wheat (Triticum aestivum L.), the Barley yellow dwarf virus (BYDV), and the BYDV vector aphid Sitobion avenae were studied under field conditions to determine the impact of these interactions on aphid populations, virus pathology and grain yield. Wheat varietal resistance to BYDV and aphids varied among the three wheat varieties studied over two consecutive years. The results demonstrated that (1) aphid peak number (APN) in the aphid + BYDV (viruliferous aphid) treatment was greater and occurred earlier than that in the non-viruliferous aphid treatment. The APN and the area under the curve of population dynamics (AUC) on a S. avenae-resistant variety 98-10-30 was significantly lower than on two aphid-susceptible varieties Tam200(13)G and Xiaoyan6. (2) The production of alatae (PA) was greater on the variety 98-10-30 than on the other varieties, and PA was greater in the aphid + BYDV treatment on 98-10-30 than in the non-viruliferous aphid treatment, but this trend was reversed on Tam200(13)G and Xiaoyan6. (3) The BYDV disease incidence (DIC) on the variety 98-10-30 was greater than that on the other two varieties in 2012, and the disease index (DID) on Tam200(13)G was lower than on the other varieties in the aphid + BYDV and BYDV treatments in 2012, but not in 2011 when aphid vector numbers were generally lower. (4) Yield loss in the aphid + BYDV treatment tended to be greater than that in the aphid or BYDV alone treatments across varieties and years. We suggested that aphid population development and BYDV transmission tend to promote each other under field conditions. The aphids + BYDV treatment caused greater yield reductions than non-viruliferous aphids or virus treatment. Wheat varietal resistance in 98-10-30 affects the aphid dispersal, virus transmission and wheat yield loss though inhibits aphid populations from increasing.
doi:10.1371/journal.pone.0106639
PMCID: PMC4153664  PMID: 25184214
23.  Effects of Electroacupuncture on Depression and the Production of Glial Cell Line–Derived Neurotrophic Factor Compared with Fluoxetine: A Randomized Controlled Pilot Study 
Abstract
Background and Objective
Postmortem studies indicate that the number and density of glial cells are reduced in different brain regions of patients with depression. Glial cell line–derived neurotrophic factor (GDNF) plays an important role in the pathogenesis of depressive disorder (DD) and might be a biomarker for damage to nerve cells. In this study, we compared the therapeutic effects of electroacupuncture (EA) and fluoxetine, a serotonin reuptake inhibitor, on DD patients, focusing on the serum level of GDNF.
Design
This was a prospective, randomized clinical trial.
Setting
Seventy-five patients with DD from the Department of Acupuncture, Beijing Hospital of Traditional Chinese Medicine, were recruited.
Intervention
Twenty patients were treated with acupuncture for 6 weeks on the acupoints of Baihui (DU20) and Zusanli (ST36). Sixteen patients were treated with acupuncture for 6 weeks on the acupoints of Taichong (LR3), Sanyinjiao (SP6), Neiguan (PC6), and Shenmen (HT7), and constituted the electroacupuncture control group. The patients received acupuncture treatment five times per week. Twenty-five patients were treated with oral fluoxetine (20 mg/day) for 6 weeks.
Outcome measures
All subjects were evaluated by the Hamilton Depression Rating Scale at four time points (0 [baseline], 2, 4, and 6 weeks after treatment). Serum GDNF was quantified in duplicate by enzyme-linked immunosorbent assay (ELISA).
Results
EA and fluoxetine had similar curative effects on DD patients. EA had a faster onset of action, better response rate, and better improvement rate than fluoxetine. Both fluoxetine and EA treatment restored the normal concentration of GDNF in the serum of DD patients.
Conclusion
EA treatment for depression is as effective as a recommended dose of fluoxetine. However, EA demonstrates an advantage in the regulation of the production of GDNF compared with fluoxetine.
doi:10.1089/acm.2011.0637
PMCID: PMC3768227  PMID: 23647408
24.  Anticancer Activities of Citrus Peel Polymethoxyflavones Related to Angiogenesis and Others 
BioMed Research International  2014;2014:453972.
Citrus is a kind of common fruit and contains multiple beneficial nutrients for human beings. Flavonoids, as a class of plant secondary metabolites, exist in citrus fruits abundantly. Due to their broad range of pharmacological properties, citrus flavonoids have gained increased attention. Accumulative in vitro and in vivo studies indicate protective effects of polymethoxyflavones (PMFs) against the occurrence of cancer. PMFs inhibit carcinogenesis by mechanisms like blocking the metastasis cascade, inhibition of cancer cell mobility in circulatory systems, proapoptosis, and antiangiogenesis. This review systematically summarized anticarcinogenic effect of citrus flavonoids in cancer therapy, together with the underlying important molecular mechanisms, in purpose of further exploring more effective use of citrus peel flavonoids.
doi:10.1155/2014/453972
PMCID: PMC4163462  PMID: 25250322
25.  Evaluation of immersion 20 MHz B-scan ultrasonography in observing lens in the alkali burn eyes 
AIM
To evaluate the accuracy of 20 MHz immersion B-scan ultrasonography in observing lens and to investigate the value of this noninvasive preoperative diagnosis method in alkali burn eyes.
METHODS
It was a comparative study. Fifty-six cases (56 eyes) of alkali burn eyes were examined by ultrasound biomicroscopy (UBM) and immersion 20 MHz B-scan ultrasonography from June 2011 to April 2013, the images were analyzed, and the ultrasonographic diagnosis compared with the operation results.
RESULTS
In 56 alkali burn eyes examined by UBM, the lens were not detected in 16 eyes; the IOL could be detected in 2 eyes; the anterior lens capsule surface or/and the front lens could be detected in 18 eyes, and lens opacification in 3 eyes of them; suspected abnormal lens were detected in the other 20 eyes. In all the same eyes examined by immersion 20 MHz B-scan ultrasonography, the lens were not detected in 16 eyes; the IOL could be detected in 2 eyes; 24 abnormal lens (opacity, lens expansion, shrinkage) and 14 normal lens were found. Compared with the intraoperative findings, the diagnostic accordance rate of the immersion 20 MHz B-scan appearance of lens was 100% (56/56), which was significantly higher than examined by UBM 57.14% (32/56) (χ2=30.55, P=0.0000).
CONCLUSION
Immersion 20 MHz B-scan ultrasonography can observe the lens accurately in alkali burn eyes. It has important clinical value to combine with UBM in eyes of alkali burn.
doi:10.3980/j.issn.2222-3959.2014.04.08
PMCID: PMC4137197  PMID: 25161933
immersion; B-scan; ultrasonography; alkali burn; ultrasound biomicroscopy

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