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1.  Impact of Osmotically Active Excipients on Bioavailability and Bioequivalence of BCS Class III Drugs 
The AAPS Journal  2013;15(4):1043-1050.
doi:10.1208/s12248-013-9509-z
PMCID: PMC3787221  PMID: 23868749
BCS; bioavailability/bioequivalence; drug absorption; excipient; osmotic potential
2.  Analysis of optimized DNase-seq reveals intrinsic bias in transcription factor footprint identification 
Nature methods  2013;11(1):73-78.
DNase-seq is a powerful technique for identifying cis-regulatory elements across the genome. We studied the key experimental parameters to optimize the performance of DNase-seq. We found that sequencing short 50-100bp fragments that accumulate in long inter-nucleosome linker regions is more efficient for identifying transcription factor binding sites than using longer fragments. We also assessed the potential of DNase-seq to predict transcription factor occupancy through the generation of nucleotide-resolution transcription factor footprints. In modeling the sequence-specific DNaseI cutting bias we found a surprisingly strong effect that varied over more than two orders of magnitude. This confounds DNaseI footprint analysis to the extent that the nucleotide resolution cleavage patterns at most transcription factor binding sites are derived from intrinsic DNaseI cleavage bias rather than from specific protein-DNA interactions. In contrast, quantitative comparison of DNaseI hypersensitivity between states can predict transcription factor occupancy associated with particular biological perturbations.
doi:10.1038/nmeth.2762
PMCID: PMC4018771  PMID: 24317252
DNaseI hypersensitivity; DNase-seq; DNaseI footprint; Chromatin dynamics; CTCF; Androgen receptor; Estrogen receptor; Transcription factor binding; Nucleosome
3.  Multiphoton imaging to identify grana, stroma thylakoid, and starch inside an intact leaf 
BMC Plant Biology  2014;14:175.
Background
Grana and starch are major functional structures for photosynthesis and energy storage of plant, respectively. Both exhibit highly ordered molecular structures and appear as micrometer-sized granules inside chloroplasts. In order to distinguish grana and starch, we used multiphoton microscopy, with simultaneous acquisition of two-photon fluorescence (2PF) and second harmonic generation (SHG) signals. SHG is sensitive to crystallized structures while 2PF selectively reveals the distribution of chlorophyll.
Result
Three distinct microstructures with different contrasts were observed, i.e. “SHG dominates”, “2PF dominates”, and “SHG collocated with 2PF”. It is known that starch and grana both emit SHG due to their highly crystallized structures, and no autofluorescence is emitted from starch, so the “SHG dominates” contrast should correspond to starch. The contrast of “SHG collocated with 2PF” is assigned to be grana, which exhibit crystallized structure with autofluorescent chlorophyll. The “2PF dominates” contrast should correspond to stroma thylakoid, which is a non-packed membrane structure with chrolophyll. The contrast assignment is further supported by fluorescence lifetime measurement.
Conclusion
We have demonstrated a straightforward and noninvasive method to identify the distribution of grana and starch within an intact leaf. By merging the 2PF and SHG images, grana, starch and stroma thylakoid can be visually distinguished. This approach can be extended to the observation of 3D grana distribution and their dynamics in living plants.
doi:10.1186/1471-2229-14-175
PMCID: PMC4104400  PMID: 24969621
Grana; Starch; Two-photon fluorescence; Second harmonic generation
4.  Cytotoxic effect of a combination of bluetongue virus and radiation on prostate cancer 
This study aimed to investigate the lethal effect of the combination of bluetongue virus (BTV) and radiation on RM-1 murine prostate cancer cells in vitro and in vivo. Various cell lines were infected with BTV and the cytotoxicity was tested by a lactate dehydrogenase (LDH) release bioassay. Additionally, the RM-1 cells were treated with radiation and/or BTV to assess cell viability using the Cell Counting Kit-8 method. The levels of apoptosis of the RM-1 cells were detected by fluorescence-activated cell sorting (FACS). To identify a possible mechanism for the radiation-induced change in the oncolytic activity of BTV, cell cycle analyses were performed. The effects of different schedules of BTV and radiotherapy on cytotoxicity were assessed in vitro and the combined effect was also assessed in tumor models in vivo. The results demonstrated that BTV had a selective cytotoxic effect on RM-1 and PC-3 cancer cells, but did not affect normal cells, specifically, human umbilical vein endothelial cells and smooth muscle cells. The combination of BTV and radiation enhanced the cytotoxicity compared with that of each agent alone and had a synergistic effect in vitro and in vivo. The results of the FACS confirmed that radiotherapy induced apoptosis, as did BTV alone, and the combination treatment generated the most prominent levels of apoptosis, which were the highest in the early stage. The analysis of the cell cycle indicated that the G2-M phase levels increased after irradiation followed by infection with BTV. In conclusion, the combination of BTV and radiotherapy had an enhanced cytotoxic effect on RM-1 cells in vitro and in vivo compared with that of either treatment alone, and demonstrated a synergistic efficacy, in addition to a marked apoptosis-inducing effect. These results support the future investigation of BTV for potential clinical use in patients with prostate cancer.
doi:10.3892/etm.2014.1751
PMCID: PMC4079398  PMID: 25009632
bluetongue virus; radiotherapy; prostate cancer; combined effects; cell apoptosis
6.  Extracellular Heat Shock Protein 90 Signals through Subdomain II and the NPVY Motif of LRP-1 Receptor to Akt1 and Akt2: a Circuit Essential for Promoting Skin Cell Migration In Vitro and Wound Healing In Vivo 
Molecular and Cellular Biology  2013;33(24):4947-4959.
Normal cells secrete heat shock protein 90 alpha (Hsp90α) in response to tissue injury. Tumor cells have managed to constitutively secrete Hsp90α during invasion and metastasis. The sole function of extracellular Hsp90α (eHsp90α) is to promote cell motility, a critical event for both wound healing and tumor progression. The mechanism of promotility action by eHsp90α, however, has remained elusive. A key issue is whether eHsp90α still acts as a chaperone outside the cells or is a new and bona fide signaling molecule. Here, we have provided evidence that eHsp90α utilizes a unique transmembrane signaling mechanism to promote cell motility and wound healing. First, subdomain II in the extracellular part of low-density lipoprotein receptor-related protein 1 (LRP-1) receives the eHsp90α signal. Then, the NPVY but not the NPTY motif in the cytoplasmic tail of LRP-1 connects eHsp90α signaling to serine 473 but not threonine 308 phosphorylation in Akt kinases. Individual knockdown of Akt1, Akt2, or Akt3 revealed the importance of Akt1 and Akt2 in eHsp90α-induced cell motility. Akt gene rescue experiments suggest that Akt1 and Akt2 work in concert, rather than independently, to mediate eHsp90α promotility signaling. Finally, Akt1 and Akt2 knockout mice showed impaired wound healing that cannot be corrected by topical application with the eHsp90α protein.
doi:10.1128/MCB.00559-13
PMCID: PMC3889557  PMID: 24126057
7.  Culture and Characterization of Microglia from the Adult Murine Retina 
The Scientific World Journal  2014;2014:894368.
Purpose. To develop a protocol for isolating and culturing murine adult retinal microglia and to characterize the phenotype and function of the cultured cells. Method. Retinal single-cell suspensions were prepared from adult MF1 mice. Culture conditions including culture medium, growth factors, seeding cell density, and purification of microglia from the mixed cultures were optimised. Cultured retinal microglial cells were phenotyped using the surface markers CD45, CD11b, and F4/80. Their ability to secrete proinflammatory cytokines in response to lipopolysaccharide (LPS) stimulation was examined using cytometric bead array (CBA) assay. Results. Higher yield was obtained when retinal single-cell suspension was cultured at the density of 0.75 × 106 cells per cm2 in Dulbecco's modified Eagle medium (DMEM)/F12 + Glutamax supplement with 20% fetal calf serum (FCS) and 20% L929 supernatant. We identified day 10 to be the optimum day of microglial isolation. Over 98% of the cells isolated were positive for CD45, CD11b, and F4/80. After stimulating with LPS they were able to secrete proinflammatory cytokines such as IL-6 and TNF-α and express CD86, CD40, and MHC-II. Conclusion. We have developed a simple method for isolating and culturing retinal microglia from adult mice.
doi:10.1155/2014/894368
PMCID: PMC4060747  PMID: 24987746
8.  Loss of Synaptic Connectivity, Particularly in Second Order Neurons Is a Key Feature of Diabetic Retinal Neuropathy in the Ins2Akita Mouse 
PLoS ONE  2014;9(5):e97970.
Retinal neurodegeneration is a key component of diabetic retinopathy (DR), although the detailed neuronal damage remains ill-defined. Recent evidence suggests that in addition to amacrine and ganglion cell, diabetes may also impact on other retinal neurons. In this study, we examined retinal degenerative changes in Ins2Akita diabetic mice. In scotopic electroretinograms (ERG), b-wave and oscillatory potentials were severely impaired in 9-month old Ins2Akita mice. Despite no obvious pathology in fundoscopic examination, optical coherence tomography (OCT) revealed a progressive thinning of the retina from 3 months onwards. Cone but not rod photoreceptor loss was observed in 3-month-old diabetic mice. Severe impairment of synaptic connectivity at the outer plexiform layer (OPL) was detected in 9-month old Ins2Akita mice. Specifically, photoreceptor presynaptic ribbons were reduced by 25% and postsynaptic boutons by 70%, although the density of horizontal, rod- and cone-bipolar cells remained similar to non-diabetic controls. Significant reductions in GABAergic and glycinergic amacrine cells and Brn3a+ retinal ganglion cells were also observed in 9-month old Ins2Akita mice. In conclusion, the Ins2Akita mouse develops cone photoreceptor degeneration and the impairment of synaptic connectivity at the OPL, predominately resulting from the loss of postsynaptic terminal boutons. Our findings suggest that the Ins2Akita mouse is a good model to study diabetic retinal neuropathy.
doi:10.1371/journal.pone.0097970
PMCID: PMC4029784  PMID: 24848689
9.  Extracellular Hsp90 (eHsp90) as the Actual Target in Clinical Trials: Intentionally or Unintentionally 
Despite extensive investigative studies and clinical trials over the past two decades, we still do not understand why cancer cells are more sensitive to the cellular toxicity of Hsp90 inhibitors than normal cells. We still do not understand why only some cancer cells are sensitive to the Hsp90 inhibitors. Based on studies of the past few years, we argue that the selected sensitivity of cancer cells to Hsp90 inhibitors, such as 17-N-allylamino-17-demethoxygeldanamycin, is due to inhibition of the extracellular Hsp90 (eHsp90) rather than intracellular Hsp90 by these inhibitors. Because not all tumor cells utilize eHsp90 for motility, invasion and metastasis, only the group of “eHsp90-dependent” cancer cells is sensitive to Hsp90 inhibitors. If these notions prove to be true, pharmaceutical agents that selectively target eHsp90 should be more effective on tumor cells and less toxic on normal cells than current inhibitors that nondiscriminatively target both extracellular and intracellular Hsp90.
doi:10.1016/B978-0-12-407697-6.00005-2
PMCID: PMC4023563  PMID: 23445811
10.  A novel genetic map of wheat: utility for mapping QTL for yield under different nitrogen treatments 
BMC Genetics  2014;15:57.
Background
Common wheat (Triticum aestivum L.) is one of the most important food crops worldwide. Wheat varieties that maintain yield (YD) under moderate or even intense nitrogen (N) deficiency can adapt to low input management systems. A detailed genetic map is necessary for both wheat molecular breeding and genomics research. In this study, an F6:7 recombinant inbred line population comprising 188 lines was used to construct a novel genetic map and subsequently to detect quantitative trait loci (QTL) for YD and response to N stress.
Results
A genetic map consisting of 591 loci distributed across 21 wheat chromosomes was constructed. The map spanned 3930.7 cM, with one marker per 6.7 cM on average. Genomic simple sequence repeat (g-SSR), expressed sequence tag-derived microsatellite (e-SSR), diversity arrays technology (DArT), sequence-tagged sites (STS), sequence-related amplified polymorphism (SRAP), and inter-simple sequence repeat (ISSR) molecular markers were included in the map. The linear relationships between loci found in the present map and in previously compiled physical maps were presented, which were generally in accordance. Information on the genetic and physical positions and allele sizes (when possible) of 17 DArT, 50 e-SSR, 44 SRAP, five ISSR, and two morphological markers is reported here for the first time. Seven segregation distortion regions (SDR) were identified on chromosomes 1B, 3BL, 4AL, 6AS, 6AL, 6BL, and 7B. A total of 22 and 12 QTLs for YD and yield difference between the value (YDDV) under HN and the value under LN were identified, respectively. Of these, QYd-4B-2 and QYddv-4B, two major stable QTL, shared support interval with alleles from KN9204 increasing YD in LN and decreasing YDDV. We probe into the use of these QTLs in wheat breeding programs. Moreover, factors affecting the SDR and total map length are discussed in depth.
Conclusions
This novel map may facilitate the use of novel markers in wheat molecular breeding programs and genomics research. Moreover, QTLs for YD and YDDV provide useful markers for wheat molecular breeding programs designed to increase yield potential under N stress.
doi:10.1186/1471-2156-15-57
PMCID: PMC4038385  PMID: 24885313
Genetic map; Molecular marker; Quantitative trait loci; Wheat; Yield
11.  Association between IL-21 gene rs907715 polymorphisms and Graves’ disease in a Southern Chinese population 
Interleukin-21 (IL-21) is a pleiotropic cytokine linking innate and adaptive immune responses, which has been reported to play a key role in multiple autoimmune diseases. The aim of the present case-control study was to investigate the genetic association between single nucleotide polymorphisms (SNPs) of rs907715 within the IL-21 gene and Graves’ disease (GD) in a Southern Chinese population. A total of 211 patients with GD and 212 control subjects were recruited for the study. IL-21 gene rs907715 polymorphisms were detected by direct DNA sequencing. The results indicated that the frequencies of the GG genotype and the G allele in GD patients were significantly increased when compared with the frequencies in the controls (P=6.7×10−3 and P=2.0×10−5, respectively). In addition, the frequency of the AA genotype was much lower in the patient group when compared with the control group (16.6 vs. 34.0%; P=4.0×10−5). Furthermore, the G allele of rs907715 was associated with relapse in GD patients. These observations indicated that polymorphisms of IL-21/rs907715 may affect the susceptibility to GD in a Southern Chinese population. The G allele was significantly associated with an increased risk of GD development, whereas the A allele may lower the susceptibility to GD.
doi:10.3892/etm.2014.1707
PMCID: PMC4061203  PMID: 24944624
interleukin-21; Graves’ disease; gene polymorphisms
12.  Toward Discovering New Anti-Cancer Agents Targeting Topoisomerase IIα: A Facile Screening Strategy Adaptable to High Throughput Platform 
PLoS ONE  2014;9(5):e97008.
Topoisomerases are a family of vital enzymes capable of resolving topological problems in DNA during various genetic processes. Topoisomerase poisons, blocking reunion of cleaved DNA strands and stabilizing enzyme-mediated DNA cleavage complex, are clinically important antineoplastic and anti-microbial agents. However, the rapid rise of drug resistance that impedes the therapeutic efficacy of these life-saving drugs makes the discovering of new lead compounds ever more urgent. We report here a facile high throughput screening system for agents targeting human topoisomerase IIα (Top2α). The assay is based on the measurement of fluorescence anisotropy of a 29 bp fluorophore-labeled oligonucleotide duplex. Since drug-stabilized Top2α-bound DNA has a higher anisotropy compared with free DNA, this assay can work if one can use a dissociating agent to specifically disrupt the enzyme/DNA binary complexes but not the drug-stabilized ternary complexes. Here we demonstrate that NaClO4, a chaotropic agent, serves a critical role in our screening method to differentiate the drug-stabilized enzyme/DNA complexes from those that are not. With this strategy we screened a chemical library of 100,000 compounds and obtained 54 positive hits. We characterized three of them on this list and demonstrated their effects on the Top2α–mediated reactions. Our results suggest that this new screening strategy can be useful in discovering additional candidates of anti-cancer agents.
doi:10.1371/journal.pone.0097008
PMCID: PMC4014593  PMID: 24809695
13.  Epidemiology of Acute Respiratory Infections in Children in Guangzhou: A Three-Year Study 
PLoS ONE  2014;9(5):e96674.
Acute Respiratory Infections (ARI) are some of the most common human diseases worldwide. However, they have a complex and diverse etiology, and the characteristics of the pathogens involved in respiratory infections in developing countries are not well understood. In this work, we analyzed the characteristics of 17 common respiratory pathogens in children (≤14 years old) with ARI in Guangzhou, southern China over a 3-year period using real-time polymerase chain reaction. Pathogens were identified in 2361/4242 (55.7%) patients, and the positivity rate varied seasonally. Ten of the 17 pathogens investigated showed positivity rates of more than 5%. The most frequently detected pathogens were respiratory syncytial virus (768/2361, 32.5%), influenza A virus (428/2361, 18.1%), enterovirus (138/2361, 13.3%), Mycoplasma pneumoniae (267/2361, 11.3%) and adenovirus (213/2361, 9.0%). Co-pathogens were common and found in 503 of 2361 (21.3%) positive samples. When ranked according to frequency of occurrence, the pattern of co-pathogens was similar to that of the primary pathogens, with the exception of human bocavirus, human coronavirus and human metapneumovirus. Significant differences were found in age prevalence in 10 of the 17 pathogens (p≤0.009): four basic patterns were observed, A: detection rates increased with age, B: detection rates declined with age, C: the detection rate showed distinct peaks or D: numbers of patients were too low to detect a trend or showed no significant difference among age groups (p>0.05). These data will be useful for planning vaccine research and control strategies and for studies predicting pathogen prevalence.
doi:10.1371/journal.pone.0096674
PMCID: PMC4010508  PMID: 24797911
14.  Development and characterization of 16 polymorphic microsatellite markers from Taiwan cow-tail fir, Keteleeria davidiana var. formosana (Pinaceae) and cross-species amplification in other Keteleeria taxa 
BMC Research Notes  2014;7:255.
Background
Keteleeria davidiana var. formosana (Pinaceae), Taiwan cow-tail fir, is an endangered species listed on the IUCN Red List of Threatened Species and only two populations remain, both on the Taiwan Island. Sixteen polymorphic microsatellite loci were developed in an endangered and endemic gymnosperm species, Keteleeria davidiana var. formosana, and were tested in an additional 6 taxa, K. davidiana var. calcarea, K. davidiana var. chienpeii, K. evelyniana, K. fortunei, K. fortunei var. cyclolepis, and K. pubescens, to evaluate the genetic variation available for conservation management and to reconstruct the phylogeographic patterns of this ancient lineage.
Findings
Polymorphic primer sets were developed from K. davidiana var. formosana using the modified AFLP and magnetic bead enrichment method. The number of alleles ranged from 3 to 16, with the observed heterozygosity ranging from 0.28 to 1.00. All of the loci were found to be interspecifically amplifiable.
Conclusions
These polymorphic and transferable loci will be potentially useful for future studies that will focus on identifying distinct evolutionary units within species and establishing the phylogeographic patterns and the process of speciation among closely related species.
doi:10.1186/1756-0500-7-255
PMCID: PMC4001355  PMID: 24755442
Keteleeria; Conservation; Microsatellites; Simple sequence repeat markers; Pinaceae
15.  Isolation and Characterization of Broad Spectrum Coaggregating Bacteria from Different Water Systems for Potential Use in Bioaugmentation 
PLoS ONE  2014;9(4):e94220.
The bridging bacteria with broad-spectrum coaggregation ability play an important role during multispecies-biofilm development. In this study, through a visual and semi-quantitative assay, twenty-two bacterial strains with aggregation ability were obtained from 8 different water environments, and these strains were assigned to 7 genera according to their 16S rDNA and they were Aeromonas, Bacillus, Comamonas, Exiguobacterium, Pseudomonas, Shewanella and Comamonas. Furthermore, all possible 231 pairwise combinations among these 22 strains were explored for coaggregation ability by spectrophotometric assay. Among all these strains, it was found that Bacillus cereus G5 and Bacillus megaterium T1 coaggregated with themajority of assayed other strains, 90.5% (19 of 21 strains) and 76.2% respectively (17 of 21 strains) at a higher coaggregation rates (A.I. greater than 50%), indicating they have a broad-spectrum coaggregation property. The images of coaggregates also confirmed the coexistence of G5 and T1 with their partner strains. Biofilm biomass development of G5 cocultured with each of its partner strains were further evaluateded. The results showed that 15 of 21 strains, when paired with G5, developed greater biofilm biomass than the monocultures. Furthermore, the images from both fluorescence microscopy and scanning electron microscopy (SEM) demonstrated that G5 and A3-GFP (a 3,5-dinitrobenzoic acid-degrading strain, staining with gfp),could develop a typical spatial structure of dual-species biofilm when cocultured. These results suggested that bridging-bacteria with a broad spectrum coaggregating ability, such as G5,could mediate the integration of exogenous degrading bacteria into biofilms and contribute to the bioaugmentation treatment.
doi:10.1371/journal.pone.0094220
PMCID: PMC3988075  PMID: 24736645
16.  Left ventricular torsional mechanics and myocardial iron load in beta-thalassaemia major: a potential role of titin degradation 
Background
Iron may damage sarcomeric proteins through oxidative stress. We explored the left ventricular (LV) torsional mechanics in patients with beta-thalassaemia major and its relationship to myocardial iron load. Using HL-1 cell and B6D2F1 mouse models, we further determined the impact of iron load on proteolysis of the giant sarcomeric protein titin.
Methods and results
In 44 thalassaemia patients aged 25 ± 7 years and 38 healthy subjects, LV torsion and twisting velocities were determined at rest using speckle tracking echocardiography. Changes in LV torsional parameters during submaximal exercise testing were further assessed in 32 patients and 17 controls. Compared with controls, patients had significantly reduced LV apical rotation, torsion, systolic twisting velocity, and diastolic untwisting velocity. T2* cardiac magnetic resonance findings correlated with resting diastolic untwisting velocity. The increments from baseline and resultant LV torsion and systolic and diastolic untwisting velocities during exercise were significantly lower in patients than controls. Significant correlations existed between LV systolic torsion and diastolic untwisting velocities in patients and controls, both at rest and during exercise. In HL-1 cells and ventricular myocardium of B6D2F1 mice overloaded with iron, the titin-stained pattern of sarcomeric structure became disrupted. Gel electrophoresis of iron-overloaded mouse myocardial tissue further showed significant decrease in the amount of titin isoforms and increase in titin degradation products.
Conclusions
Resting and dynamic LV torsional mechanics is impaired in patients with beta-thalassaemia major. Cell and animal models suggest a potential role of titin degradation in iron overload-induced alteration of LV torsional mechanics.
doi:10.1186/1471-2261-14-49
PMCID: PMC4021258  PMID: 24725620
Thalassaemia; Ventricular rotation; Ventricular mechanics; Exercise echocardiography; Titin
17.  Characterization of Imidazoline Receptors in Blood Vessels for the Development of Antihypertensive Agents 
BioMed Research International  2014;2014:182846.
It has been indicated that activation of peripheral imidazoline I2-receptor (I-2R) may reduce the blood pressure in spontaneously hypertensive rats (SHRs). Also, guanidinium derivatives show the ability to activate imidazoline receptors. Thus, it is of special interest to characterize the I-2R using guanidinium derivatives in blood vessels for development of antihypertensive agent(s). Six guanidinium derivatives including agmatine, amiloride, aminoguanidine, allantoin, canavanine, and metformin were applied in this study. Western blot analysis was used for detecting the expression of imidazoline receptor in tissues of Wistar rats. The isometric tension of aortic rings isolated from male rats was also estimated. The expression of imidazoline receptor on rat aorta was identified. However, guanidinium derivatives for detection of aortic relaxation were not observed except agmatine and amiloride which induced a marked relaxation in isolated aortic rings precontracted with phenylephrine or KCl. Both relaxations induced by agmatine and amiloride were attenuated by glibenclamide at concentration enough to block ATP-sensitive potassium (KATP) channels. Meanwhile, only agmatine-induced relaxation was abolished by BU224, a selective antagonist of imidazoline I2-receptors. Taken together, we suggest that agmatine can induce vascular relaxation through activation of peripheral imidazoline I2-receptor to open KATP channels. Thus, agmatine-like compound has the potential to develop as a new therapeutic agent for hypertension in the future.
doi:10.1155/2014/182846
PMCID: PMC3996295  PMID: 24800210
18.  Intravitreal Injection of Normal Saline Induces Retinal Degeneration in the C57BL/6J Mouse 
Purpose
To investigate the adverse effect of intravitreal injection of normal saline (NS) and phosphate buffered saline (PBS) in mouse eyes.
Methods
NS or PBS was injected intravitreally into C57BL/6J mouse eyes. Retinal lesions were monitored by fundus imaging, spectral-domain optical coherence tomography (SD-OCT), and histological investigations. Retinal immune gene expression was determined by real-time polymerase chain reaction (PCR). The toxic effect of NS and PBS or retinal protein from NS- or PBS-injected eyes on retinal pigment epithelium (RPE) was tested in B6-RPE-07 mouse RPE cell cultures.
Results
Intravitreal injection of NS dose-dependently induced localized retinal lesion in mice. Histological investigations revealed multiple vacuoles in photoreceptor outer segments and RPE cells. The lesions recovered over time and by 3 weeks post injection the majority of lesions vanished in eyes receiving 1 μl NS. Inflammatory genes, including TNF-α, IL-1β, IL-6, iNOS, and VEGF were upregulated in NS injected eyes. Intravitreal injection of PBS did not cause any pathology. The treatment of B6-RPE07 cells with 30% PBS or 30% NS did not affect RPE viability. However, incubation of 1-μg/ml retinal protein from NS-injected eyes, but not PBS-injected eyes induced RPE cell death.
Conclusion
NS is toxic to the C57BL/6J mouse retina and should not be used as a vehicle for intraocular injection. PBS is not toxic to the retina and is a preferred vehicle.
Translational Relevance
NS is not a physiological solution for intraocular injection in the C57BL/6J mice and questions its suitability for intraocular injection in other species, including human.
doi:10.1167/tvst.3.2.3
PMCID: PMC3969215  PMID: 24757593
intraocular injection; retinal pigment epithelium; toxicity; photoreceptors; saline
19.  Antihypertensive Action of Allantoin in Animals 
BioMed Research International  2014;2014:690135.
The agonists of imidazoline I-1 receptors (I-1R) are widely used to lower blood pressure. It has been indicated that guanidinium derivatives show an ability to activate imidazoline receptors. Also, allantoin has a chemical stricture similar to guanidinium derivatives. Thus, it is of special interest to characterize the effect of allantoin on I-1R. In conscious male spontaneous hypertensive rats (SHRs), mean blood pressure (MBP) was recorded using the tail-cuff method. Furthermore, the hemodynamic analyses in catheterized rats were applied to measure the actions of allantoin in vivo. Allantoin decreased blood pressures in SHRs at 30 minutes, as the most effective time. Also, this antihypertensive action was shown in a dose-dependent manner from SHRs treated with allantoin. Moreover, in anesthetized rats, allantoin inhibited cardiac contractility and heart rate as showing in hemodynamic dP/dt max significantly. Also, the peripheral blood flow was markedly increased by allantoin. Both actions were diminished by efaroxan at the dose sufficient to block I-1R. Thus, we suggest that allantoin, as I-1R agonist, has the potential to develop as a new therapeutic agent for hypertension in the future.
doi:10.1155/2014/690135
PMCID: PMC3972943  PMID: 24745022
20.  RAGE Regulates Immune Cell Infiltration and Angiogenesis in Choroidal Neovascularization 
PLoS ONE  2014;9(2):e89548.
Purpose
RAGE regulates pro-inflammatory responses in diverse cells and tissues. This study has investigated if RAGE plays a role in immune cell mobilization and choroidal neovascular pathology that is associated with the neovascular form of age-related macular degeneration (nvAMD).
Methods
RAGE null (RAGE−/−) mice and age-matched wild type (WT) control mice underwent laser photocoagulation to generate choroidal neovascularization (CNV) lesions which were then analyzed for morphology, S100B immunoreactivity and inflammatory cell infiltration. The chemotactic ability of bone marrow derived macrophages (BMDMs) towards S100B was investigated.
Results
RAGE expression was significantly increased in the retina during CNV of WT mice (p<0.001). RAGE−/− mice exhibited significantly reduced CNV lesion size when compared to WT controls (p<0.05). S100B mRNA was upregulated in the lasered WT retina but not RAGE−/− retina and S100B immunoreactivity was present within CNV lesions although levels were less when RAGE−/− mice were compared to WT controls. Activated microglia in lesions were considerably less abundant in RAGE−/− mice when compared to WT counterparts (p<0.001). A dose dependent chemotactic migration was observed in BMDMs from WT mice (p<0.05–0.01) but this was not apparent in cells isolated from RAGE−/− mice.
Conclusions
RAGE-S100B interactions appear to play an important role in CNV lesion formation by regulating pro-inflammatory and angiogenic responses. This study highlights the role of RAGE in inflammation-mediated outer retinal pathology.
doi:10.1371/journal.pone.0089548
PMCID: PMC3935881  PMID: 24586862
21.  α-Tocopherol protects keratinocytes against ultraviolet A irradiation by suppressing glutathione depletion, lipid peroxidation and reactive oxygen species generation 
Biomedical Reports  2014;2(3):419-423.
This study aimed to investigate whether α-tocopherol is able to protect keratinocytes against ultraviolet A (UVA) radiation by increasing glutathione (γ-glutamylcysteinylglycine; GSH) levels or decreasing lipid peroxidation and reactive oxygen species (ROS) generation. The cell survival fraction was 43.6% when keratinocytes were irradiated with UVA at a dose of 8 J/cm2. α-Tocopherol was added prior to UVA irradiation and the cell viability was assayed. The cell survival fractions were 60.2, 77.1, 89.0, 92.9 and 96.2% when α-tocopherol was added at concentrations of 2.9, 5.9, 8.8, 11.8 and 14.7 IU/ml, respectively. These results suggested that α-tocopherol is capable of protecting keratinocytes against UVA irradiation. Furthermore, the levels of GSH, lipid peroxidation and ROS were measured. The levels of GSH were 0.354 and 0.600 mmol/g protein in keratinocytes irradiated with UVA (8 J/cm2) and in non-irradiated cells, respectively, whereas they were 0.364, 0.420, 0.525, 0.540 and 0.545 mmol/g protein when α-tocopherol was added at concentrations of 2.9, 5.9, 8.8, 11.8 and 14.7 IU/ml, respectively. The levels of lipid peroxidation were 20.401 or 5.328 μmol/g [malondialdehyde (MDA)/protein] in keratinocytes irradiated with UVA (8 J/cm2) and in non-irradiated cells, respectively, whereas they were 11.685, 6.544, 5.847, 4.390 and 2.164 μmol/g (MDA/protein) when α-tocopherol was added at concentrations of 2.9, 5.9, 8.8, 11.8 and 14.7 IU/ml, respectively. The levels of ROS were 3,952.17 or 111.87 1/mg protein in keratinocytes irradiated with UVA (8 J/cm2) and in non-irradiated cells, respectively, whereas they were 742.48, 579.36, 358.16, 285.63 and 199.82 1/mg protein when α-tocopherol was added at concentrations of 2.9, 5.9, 8.8, 11.8 and 14.7 IU/ml, respectively. These findings suggested that α-tocopherol protects keratinocytes against UVA irradiation, possibly through increasing the levels of GSH or decreasing the levels of lipid peroxidation and ROS generation.
doi:10.3892/br.2014.236
PMCID: PMC3990220  PMID: 24748986
α-tocopherol; keratinocytes; ultraviolet A radiation; glutathione; lipid peroxidation
22.  Validation of the Antidiabetic and Hypolipidemic Effects of Clitocybe nuda by Assessment of Glucose Transporter 4 and Gluconeogenesis and AMPK Phosphorylation in Streptozotocin-Induced Mice 
The study was designed to investigate the effects of extract of Clitocybe nuda (CNE) on type 1 diabetes mellitus and dyslipidemia in streptozotocin- (STZ-) induced diabetic mice. Diabetes was induced by injection of STZ. Diabetic mice were randomly divided into five groups and given orally CNE (C1: 0.2, C2: 0.5, and C3: 1.0 g/kg body weight) or metformin (Metf) or vehicle for 4 weeks. STZ induction decreased in the levels of insulin, body weight, and the weight of skeletal muscle, whereas the levels of blood glucose, hemoglobin nonenzymatically (percent HbA1c), and circulating triglyceride (P < 0.001, P < 0.001, and P < 0.01, resp.) were increased. CNE decreased the levels of blood glucose, HbA1c, and triglyceride levels, whereas it increased the levels of insulin and leptin compared with the vehicle-treated STZ group. STZ induction caused a decrease in the protein contents of skeletal muscular and hepatic phosphorylation of AMP-activated protein kinase (phospho-AMPK) and muscular glucose transporter 4 (GLUT4). Muscular phospho-AMPK contents were increased in C2-, C3-, and Metf-treated groups. CNE and Metf significantly increased the muscular proteins of GLUT4. Liver phospho-AMPK showed an increase in all CNE- and Metf-treated groups combined with the decreased hepatic glucose production by decreasing phosphenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase), and 11beta hydroxysteroid dehydroxygenase (11β-HSD1) gene, which contributed to attenuating diabetic state. The study indicated that the hypoglycemic properties of CNE were related to both the increased muscular glucose uptake and the reduction in hepatic gluconeogenesis. CNE exerts hypolipidemic effect by increasing gene expressions of peroxisome proliferator-activated receptor α (PPARα) and decreasing expressions of fatty acid synthesis, including acyl-coenzyme A: diacylglycerol acyltransferase (DGAT) 2. Therefore, amelioration of diabetic and dyslipidemic state by CNE in STZ-induced diabetic mice occurred by regulation of GLUT4, PEPCK, DGAT2, and AMPK phosphorylation.
doi:10.1155/2014/705636
PMCID: PMC3930086  PMID: 24639883
23.  Autocrine CCL3 and CCL4 Induced by the Oncoprotein LMP1 Promote Epstein-Barr Virus-Triggered B Cell Proliferation 
Journal of Virology  2013;87(16):9041-9052.
Epstein-Barr virus (EBV) alters the regulation and expression of a variety of cytokines in its host cells to modulate host immune surveillance and facilitate viral persistence. Using cytokine antibody arrays, we found that, in addition to the cytokines reported previously, two chemotactic cytokines, CCL3 and CCL4, were induced in EBV-infected B cells and were expressed at high levels in all EBV-immortalized lymphoblastoid cell lines (LCLs). Furthermore, EBV latent membrane protein 1 (LMP1)-mediated Jun N-terminal protein kinase activation was responsible for upregulation of CCL3 and CCL4. Inhibition of CCL3 and CCL4 in LCLs using a short hairpin RNA approach or by neutralizing antibodies suppressed cell proliferation and caused apoptosis, indicating that autocrine CCL3 and CCL4 are required for LCL survival and growth. Importantly, significant amounts of CCL3 were detected in EBV-positive plasma from immunocompromised patients, suggesting that EBV modulates this chemokine in vivo. This study reveals the regulatory mechanism and a novel function of CCL3 and CCL4 in EBV-infected B cells. CCL3 might be useful as a therapeutic target in EBV-associated lymphoproliferative diseases and malignancies.
doi:10.1128/JVI.00541-13
PMCID: PMC3754032  PMID: 23760235
24.  Casein Kinase 1 Epsilon Expression Predicts Poorer Prognosis in Low T-Stage Oral Cancer Patients 
Casein kinase 1 is a group of ubiquitous serine/threonine kinases that are involved in normal cellular functions and several pathological conditions, such as DNA repair, cell cycle progression, cytokinesis, differentiation, and apoptosis. Recent studies have indicated that casein kinase 1-epsilon (CK1ɛ) and casein kinase 1-delta (CK1δ) expression has a role in human cancers. We investigated the associations between CK1ɛ and CK1δ expression and the clinical parameters of oral cancer using immunohistochemical study methods on oral squamous cell carcinoma specimens. The results of our immunohistochemical analysis showed that the loss of CK1ɛ expression was greatly associated with a poor four-year survival rate in oral cancer patients (p = 0.002). A Kaplan-Meier analysis showed that patients who had a loss of CK1ɛ expression had a considerably poorer overall survival rate than patients who had positive CK1ɛ expressions (p = 0.022). A univariate analysis revealed that patients who had a loss of CK1ɛ expression had considerably poorer overall survival (OS) than patients who had positive expression (p = 0.024, hazard ratio (HR) = 1.7). In conclusion, our data indicated that the loss of cytoplasmic CK1ɛ expression is greatly associated with poor survival and might be an adverse survival factor.
doi:10.3390/ijms15022876
PMCID: PMC3958887  PMID: 24557581
casein kinase 1 epsilon (CK1ɛ); oral cancer; overall survival
25.  Antidiabetic and Antihyperlipidemic Effects of Clitocybe nuda on Glucose Transporter 4 and AMP-Activated Protein Kinase Phosphorylation in High-Fat-Fed Mice 
The objective of this study was to evaluate the antihyperlipidemic and antihyperglycemic effects and mechanism of the extract of Clitocybe nuda (CNE), in high-fat- (HF-) fed mice. C57BL/6J was randomly divided into two groups: the control (CON) group was fed with a low-fat diet, whereas the experimental group was fed with a HF diet for 8 weeks. Then, the HF group was subdivided into five groups and was given orally CNE (including C1: 0.2, C2: 0.5, and C3: 1.0 g/kg/day extracts) or rosiglitazone (Rosi) or vehicle for 4 weeks. CNE effectively prevented HF-diet-induced increases in the levels of blood glucose, triglyceride, insulin (P < 0.001, P < 0.01, P < 0.05, resp.) and attenuated insulin resistance. By treatment with CNE, body weight gain, weights of white adipose tissue (WAT) and hepatic triacylglycerol content were reduced; moreover, adipocytes in the visceral depots showed a reduction in size. By treatment with CNE, the protein contents of glucose transporter 4 (GLUT4) were significantly increased in C3-treated group in the skeletal muscle. Furthermore, CNE reduces the hepatic expression of glucose-6-phosphatase (G6Pase) and glucose production. CNE significantly increases protein contents of phospho-AMP-activated protein kinase (AMPK) in the skeletal muscle and adipose and liver tissues. Therefore, it is possible that the activation of AMPK by CNE leads to diminished gluconeogenesis in the liver and enhanced glucose uptake in skeletal muscle. It is shown that CNE exhibits hypolipidemic effect in HF-fed mice by increasing ATGL expression, which is known to help triglyceride to hydrolyze. Moreover, antidiabetic properties of CNE occurred as a result of decreased hepatic glucose production via G6Pase downregulation and improved insulin sensitization. Thus, amelioration of diabetic and dyslipidemic states by CNE in HF-fed mice occurred by regulation of GLUT4, G6Pase, ATGL, and AMPK phosphorylation.
doi:10.1155/2014/981046
PMCID: PMC3914338  PMID: 24550994

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