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1.  Effect of 2, 5-Substituents on the Stability of Cyclic Nitrone Superoxide Spin Adducts: A Density Functional Theory Approach 
Free radical research  2010;44(7):751-778.
To design efficient spin traps for superoxide radicals, interest in the elucidation of substituent effects on the stability of superoxide spin adducts has become a necessary priority. In the present study, five cyclic nitrone superoxide spin adducts, i.e. DMPO-OOH, M3PO-OOH, EMPO-OOH, DEPMPO-OOH, and DEPDMPO-OOH, were chosen as model compounds to investigate the effect of 2,5-subsitituents on their stability, through structural analysis and decay thermodynamics using density functional theory (DFT) calculations. Analysis of the optimized geometries reveals that none of the previously proposed stabilizing factors, including intramolecular H-bonds, intramolecular nonbonding interactions, bulky steric protection, nor the C(2)–N(1) bond distance can be used to clearly explain the effect of 2,5-substituents on the stability of the spin adducts. Additionally the effect of the 2,5-substituents on the stability of the superoxide spin adducts cannot be simply clarified by Milliken charges on both atoms (nitroxyl nitrogen and nitroxyl oxygen). Subsequent study found that spin densities on the nitroxyl nitrogen and oxygen are well correlated with the half-life times of the spin adducts, and consequently are the proper parameters to characterize the effect of 2,5-substituents on their stability. Examination of the decomposition thermodynamics further supports the effect of the substituents on the persistence of cyclic nitrone superoxide spin adducts.
PMCID: PMC3074479  PMID: 20370568
2.  Intrinsic dynamics induce global symmetry in network controllability 
Scientific Reports  2015;5:8422.
Controlling complex networked systems to desired states is a key research goal in contemporary science. Despite recent advances in studying the impact of network topology on controllability, a comprehensive understanding of the synergistic effect of network topology and individual dynamics on controllability is still lacking. Here we offer a theoretical study with particular interest in the diversity of dynamic units characterized by different types of individual dynamics. Interestingly, we find a global symmetry accounting for the invariance of controllability with respect to exchanging the densities of any two different types of dynamic units, irrespective of the network topology. The highest controllability arises at the global symmetry point, at which different types of dynamic units are of the same density. The lowest controllability occurs when all self-loops are either completely absent or present with identical weights. These findings further improve our understanding of network controllability and have implications for devising the optimal control of complex networked systems in a wide range of fields.
PMCID: PMC4325315  PMID: 25672476
3.  Single siRNA Nanocapsules for Enhanced RNAi Delivery 
Journal of the American Chemical Society  2012;134(33):13542-13545.
Synthetic siRNA has been considered as a highly promising therapeutic agent for human diseases. However, clinical use of siRNA has been hampered by instability in the body and inability to deliver sufficient RNA interference compounds to the tissues or cells. To address this challenge, we present here a single siRNA nanocapsule delivery technology, which is achieved by encapsulating a single siRNA molecule within a degradable polymer nanocapsule with a diameter around 20 nm and positive surface charge. As proof-of-concept, since CCR5 is considered a major silencing target of HIV therapy, CCR5–siRNA nanocapsules were delivered into 293T cells and successfully downregulated the CCR5 RNA fused with mCherry reporter RNA. In the absence of human serum, nanocapsules and lipofectamine silenced expression of CCR5–mCherry expression to 8% and 15%, respectively. Such nanocapsules maintain the integrity of siRNA inside even after incubation with ribonuclease and serum for 1 h; under the same conditions, siRNA is degraded in the native form or when formulated with lipofectamine. In the presence of serum, CCR5–siRNA nanocapsules knocked down CCR5–mCherry expression to less than 15% while siRNAs delivered through lipofectamine slightly knocked down the expression to 55%. In summary, this work provides a novel platform for siRNA delivery that can be developed for therapeutic purposes.
PMCID: PMC4318836  PMID: 22866878
4.  Antennal Transcriptome Analysis and Comparison of Chemosensory Gene Families in Two Closely Related Noctuidae Moths, Helicoverpa armigera and H. assulta 
PLoS ONE  2015;10(2):e0117054.
To better understand the olfactory mechanisms in the two lepidopteran pest model species, the Helicoverpa armigera and H. assulta, we conducted transcriptome analysis of the adult antennae using Illumina sequencing technology and compared the chemosensory genes between these two related species. Combined with the chemosensory genes we had identified previously in H. armigera by 454 sequencing, we identified 133 putative chemosensory unigenes in H. armigera including 60 odorant receptors (ORs), 19 ionotropic receptors (IRs), 34 odorant binding proteins (OBPs), 18 chemosensory proteins (CSPs), and 2 sensory neuron membrane proteins (SNMPs). Consistent with these results, 131 putative chemosensory genes including 64 ORs, 19 IRs, 29 OBPs, 17 CSPs, and 2 SNMPs were identified through male and female antennal transcriptome analysis in H. assulta. Reverse Transcription-PCR (RT-PCR) was conducted in H. assulta to examine the accuracy of the assembly and annotation of the transcriptome and the expression profile of these unigenes in different tissues. Most of the ORs, IRs and OBPs were enriched in adult antennae, while almost all the CSPs were expressed in antennae as well as legs. We compared the differences of the chemosensory genes between these two species in detail. Our work will surely provide valuable information for further functional studies of pheromones and host volatile recognition genes in these two related species.
PMCID: PMC4319919  PMID: 25659090
7.  Intracellular CD24 disrupts the ARF–NPM interaction and enables mutational and viral oncogene-mediated p53 inactivation 
Nature Communications  2015;6:5909.
CD24 is overexpressed in nearly 70% human cancers, whereas TP53 is the most frequently mutated tumour-suppressor gene that functions in a context-dependent manner. Here we show that both targeted mutation and short hairpin RNA (shRNA) silencing of CD24 retard the growth, progression and metastasis of prostate cancer. CD24 competitively inhibits ARF binding to NPM, resulting in decreased ARF, increase MDM2 and decrease levels of p53 and the p53 target p21/CDKN1A. CD24 silencing prevents functional inactivation of p53 by both somatic mutation and viral oncogenes, including the SV40 large T antigen and human papilloma virus 16 E6-antigen. In support of the functional interaction between CD24 and p53, in silico analyses reveal that TP53 mutates at a higher rate among glioma and prostate cancer samples with higher CD24 mRNA levels. These data provide a general mechanism for functional inactivation of ARF and reveal an important cellular context for genetic and viral inactivation of TP53.
P53 is a tumour suppressor that is frequently mutated or downregulated in cancer. Here, Wang et al. show that CD24, a molecule frequently overexpressed in cancer, promotes p53 degradation by disrupting a regulatory ARF–MDM2 interaction, and silencing CD24 prevents the downregulation of p53.
PMCID: PMC4300525  PMID: 25600590
8.  The Impact of Winter Heating on Air Pollution in China 
PLoS ONE  2015;10(1):e0117311.
Fossil-fuel combustion related winter heating has become a major air quality and public health concern in northern China recently. We analyzed the impact of winter heating on aerosol loadings over China using the MODIS-Aqua Collection 6 aerosol product from 2004–2012. Absolute humidity (AH) and planetary boundary layer height (PBL) -adjusted aerosol optical depth (AOD*) was constructed to reflect ground-level PM2.5 concentrations. GIS analysis, standard statistical tests, and statistical modeling indicate that winter heating is an important factor causing increased PM2.5 levels in more than three-quarters of central and eastern China. The heating season AOD* was more than five times higher as the non-heating season AOD*, and the increase in AOD* in the heating areas was greater than in the non-heating areas. Finally, central heating tend to contribute less to air pollution relative to other means of household heating.
PMCID: PMC4309400  PMID: 25629878
9.  Extraordinarily low evolutionary rates of short wavelength-sensitive opsin pseudogenes 
Gene  2013;534(1):10.1016/j.gene.2013.09.114.
Aquatic organisms such as cichlids, coelacanths, seals, and cetaceans are active in UV-blue color environments, but many of them mysteriously lost their abilities to detect these colors. The loss of these functions is a consequence of the pseudogenization of their short wavelength-sensitive (SWS1) opsin genes without gene duplication. We show that the SWS1 gene (BdenS1ψ) of the deep-sea fish, pearleye (Benthalbella dentata), became a pseudogene in a similar fashion about 130 million years ago (Mya) yet it is still transcribed. The rates of nucleotide substitution (~1.4 × 10−9 /site/year) of the pseudogenes of these aquatic species as well as some prosimian and bat species are much smaller than the previous estimates for the globin and immunoglobulin pseudogenes.
PMCID: PMC3852691  PMID: 24125953
Aquatic animals; SWS1 pseudogenes; Molecular evolution
10.  Arsenite binding-induced zinc loss from PARP-1 is equivalent to zinc deficiency in reducing PARP-1 activity, leading to inhibition of DNA repair 
Toxicology and applied pharmacology  2013;274(2):313-318.
Inhibition of DNA repair is a recognized mechanism for arsenic enhancement of ultraviolet radiation-induced DNA damage and carcinogenesis. Poly(ADP-ribose) polymerase-1 (PARP-1), a zinc finger DNA repair protein, has been identified as a sensitive molecular target for arsenic. The zinc finger domains of PARP-1 protein function as a critical structure in DNA recognition and binding. Since cellular poly(ADP-ribosyl)ation capacity has been positively correlated with zinc status in cells, we hypothesize that arsenite binding-induced zinc loss from PARP-1 is equivalent to zinc deficiency in reducing PARP-1 activity, leading to inhibition of DNA repair. To test this hypothesis, we compared the effects of arsenite exposure with zinc deficiency, created by using the membrane-permeable zinc chelator TPEN, on 8-OHdG formation, PARP-1 activity and zinc binding to PARP-1 in HaCat cells. Our results show that arsenite exposure and zinc deficiency had similar effects on PARP-1 protein, whereas supplemental zinc reversed these effects. To investigate the molecular mechanism of zinc loss induced by arsenite, ICP-AES, near UV spectroscopy, fluorescence, and circular dichroism spectroscopy were utilized to examine arsenite binding and occupation of a peptide representing the first zinc finger of PARP-1. We found that arsenite binding as well as zinc loss altered the conformation of zinc finger structure which functionally leads to PARP-1 inhibition. These findings suggest that arsenite binding to PARP-1 protein created similar adverse biological effects as zinc deficiency, which establishes the molecular mechanism for zinc supplementation as a potentially effective treatment to reverse the detrimental outcomes of arsenic exposure.
PMCID: PMC3891920  PMID: 24275069
zinc deficiency; arsenite; PARP-1; DNA damage repair; zinc finger
11.  Quantum Dots Encapsulated within Phospholipid Membranes: Phase-Dependent Structure, Photostability, and Site-Selective Functionalization 
Lipid vesicle encapsulation is an efficient approach to transfer quantum dots (QDs) into aqueous solutions, which is important for renewable energy applications and biological imaging. However, little is known about the molecular organization at the interface between a QD and lipid membrane. To address this issue, we investigated the properties of 3.0 nm CdSe QDs encapsulated within phospholipid membranes displaying a range of phase transition temperatures (Tm). Theoretical and experimental results indicate that the QD locally alters membrane structure, and in turn, the physical state (phase) of the membrane controls the optical and chemical properties of the QDs. Using photoluminescence, ICP-MS, optical microscopy, and ligand exchange studies, we found that the Tm of the membrane controls optical and chemical properties of lipid vesicle-embedded QDs. Importantly, QDs encapsulated within gel-phase membranes were ultrastable, providing the most photostable non-core/shell QDs in aqueous solution reported to date. Atomistic molecular dynamics simulations support these observations and indicate that membranes are locally disordered displaying greater disordered organization near the particle–solution interface. Using this asymmetry in membrane organization near the particle, we identify a new approach for site-selective modification of QDs by specifically functionalizing the QD surface facing the outer lipid leaflet to generate gold nanoparticle–QD assemblies programmed by Watson–Crick base-pairing.
PMCID: PMC3985776  PMID: 24417287
12.  Genome Sequence of Thermoactinomyces daqus H-18, a Novel Thermophilic Species Isolated from High-Temperature Daqu 
Genome Announcements  2015;3(1):e01394-14.
Thermoactinomyces daqus H-18 is a new species of Thermoactinomyces isolated from high-temperature Daqu used in the fermentation of Bandongjing sesame-flavor liquor. Its genome was sequenced and assembled (3.44 Mb). The coding sequences (CDSs) that correlated to high-temperature tolerance were annotated. The metabolic pathways for the compounds responsible for flavor were also found.
PMCID: PMC4290991  PMID: 25573938
13.  The Relationship Between Disability-Adjusted Life Years of Cataracts and Ambient Erythemal Ultraviolet Radiation in China 
Journal of Epidemiology  2015;25(1):57-65.
Cataracts are one of the major public health problems worldwide. Ultraviolet radiation (UVR) is one of the risk factors for cataract development. We analyzed the relationship between disability-adjusted life year (DALY) rates of cataracts and UVR exposure in China.
DALY rates of cataracts and UVR exposure in 31 regions of China were calculated based on data from the Second China National Sample Survey on Disability and the United States’ National Aeronautics and Space Administration database. The relationship between the DALY rates of cataracts and UVR was estimated by Spearman rank correlation analysis and linear regression analysis.
The elderly (≥65 years) had higher DALY rates of cataracts than the whole population. The DALY rate of cataracts in the agricultural population was higher than that observed in the non-agricultural population. The DALY rates of cataracts were positively associated with UVR The DALY rates of cataracts in regions with higher UVR were higher than those in regions with lower UVR. An increase in the daily ambient erythemal UVR of 1000 J/m2 was associated with an increase in the DALY rates of cataracts by 92 DALYs/100 000 (R2 = 0.676) among the whole population, 34 DALYs/100 000 among the population <65 years old (R2 = 0.423), 607 DALYs/100 000 among the population aged 65–74 years (R2 = 0.617), and by 1342 DALYs/100 000 among the population ≥75 years old (R2 = 0.758).
DALY rates of cataracts increased with increases in UVR exposure in 31 regions of China. Greater exposure to UVR increases the disease burden of cataracts in the whole population, especially in the elderly and among the agricultural population.
PMCID: PMC4275439
cataract; UVR exposure; disease burden; DALYs
14.  A VISTA on PD-1H 
The Journal of Clinical Investigation  2014;124(5):1891-1893.
Three years ago, two research groups independently identified a previously undescribed T cell cosignaling molecule; one referred to it as V-domain Ig suppressor of T cell activation (VISTA), and the other used the term programmed death-1 homolog (PD-1H). Recombinant and ectopically expressed PD-1H functions as a coinhibitory ligand for T cell responses. However, the function of endogenous PD-1H is not clear. In this issue of the JCI, Flies and colleagues demonstrate that endogenous PD-1H on both T cells and APCs serves as a coinhibitory molecule for T cell activation and provide further support for targeting PD-1H as a therapeutic strategy for transplantation and cancers.
PMCID: PMC4001562  PMID: 24743142
15.  Decrease of Let-7f in Low-Dose Metronomic Paclitaxel Chemotherapy Contributed to Upregulation of Thrombospondin-1 in Breast Cancer 
Low-dose metronomic (LDM) paclitaxel therapy displayed a stronger anti-angiogenic activity on breast tumors with fewer side effects. Upregulation of anti-angiogenic factor Thrombospondin-1 (TSP-1) accords for therapeutic potency of LDM paclitaxel, but its molecular mechanism has not been elucidated yet. microRNAs (miRNAs) have emerged as new important regulators of tumor growth and metastasis. Here, we hypothesize that miRNAs are involved in TSP-1 overexpression in paclitaxel LDM therapy of breast tumors. The miRNA profile of tumor tissues from control, LDM and MTD groups in 4T1 mouse breast cancer model was detected by microarray, and then verified by quantitative real-time PCR (qRT-PCR). Luciferase assay and western blot were employed to explore the mechanisms of miRNAs involved in this process. We found that let-7f, let-7a, miR-19b and miR-340-5p were reduced by >2 fold, and miR-543* and miR-684 were upregulated by at least 50% in paclitaxel LDM therapy. qRT-PCR verification revealed that let-7f level was reduced most significantly in LDM therapy. Computational prediction using TargetScan and miRanda suggested THBS1 which encodes TSP-1 as a potential target for let-7f. Luciferase activity assay further confirmed that let-7f may bind to 3'UTR of THBS1 gene and inhibit its activity. Moreover, forced expression of let-7f led to a decrease of TSP-1 at both mRNA and protein levels in MCF-7 cells. Contrastly, let-7f inhibition induced an increased expression of THBS1 mRNA and TSP-1 protein, but did not affect the proliferation and apoptosis of MCF-7 cells. Paclitaxel LDM therapy led to a decrease of let-7f and the elevation of TSP-1 protein expression in MCF-7 cells, while overexpression of let-7f may abolish LDM-induced the upregulation of TSP-1 in MCF-7 cells. In summary, let-7f inhibition contributed to the upregulation of TSP-1 in paclitaxel LDM therapy, independently of proliferation, cell cycle arrest and apoptosis of breast cancer. This study indicates let-7f as a potential therapeutic target for breast tumor.
PMCID: PMC4278254  PMID: 25552929
let-7f; Thrombospondin-1; Low-dose metronomic paclitaxel therapy.
16.  Increased ARPP-19 Expression Is Associated with Hepatocellular Carcinoma 
The cAMP-regulated phosphoprotein 19 (ARPP-19) plays a key role in cell mitotic G2/M transition. Expression of ARPP-19 was increased in human hepatocellular carcinoma (HCC) compared to adjacent non-tumorous liver tissues in 36 paired liver samples, and the level of ARPP-19 in HCC tissues was positively correlated with the tumor size. To determine the interrelationship between ARPP-19 expression and HCC, we silenced ARPP-19 expression in the human hepatocarcinoma HepG2 and SMMC-7721 cells using lentivirus encoding ARPP-19 siRNA. HepG2 and SMMC-7721 cells with ARPP-19 knockdown displayed lowered cell growth rate, retarded colony formation and increased arrest at the G2/M phase transition. Silencing ARPP-19 in HCC cells resulted in decreased protein levels of phospho-(Ser) CDKs substrates and increased levels of inactivated cyclin division cycle 2 (Cdc2). Therefore, ARPP-19 may play a role in HCC pathogenesis through regulating cell proliferation.
PMCID: PMC4307242  PMID: 25547487
hepatocellular carcinoma; ARPP-19; cell proliferation; cell cycle
17.  MicroRNA-33b Suppresses Migration and Invasion by Targeting c-Myc in Osteosarcoma Cells 
PLoS ONE  2014;9(12):e115300.
MicroRNAs have emerged as fundamental regulators in gene expression through silencing gene expression at the post-transcriptional and translational levels. Osteosarcoma is the most common type of primary malignant bone tumor and is characterized by complex genetic changes and resistance to conventional treatments. In our study, the role of miR-33b in the progression and metastasis of osteosarcoma was investigated. Our results showed that miR-33b was significantly downregulated in osteosarcoma tissue and cell lines. Overexpression of miR-33b significantly inhibited cell proliferation, migration, and invasion in the MG-63 osteosarcoma cell line. Moreover, we also showed that c-Myc was negatively regulated by miR-33b at the posttranscriptional level, via a specific target site within the 3′UTR. Overexpression of c-Myc impaired miR-33b-induced inhibition of proliferation and invasion in osteosarcoma cells. The expression of c-Myc was frequently downregulated in osteosarcoma tumors and cell lines and was inversely correlated with miR-33b expression. Thus, our findings suggest that miR-33b inhibits osteosarcoma cells migration and invasion by targeting the c-Myc gene, acting as tumor suppressor. The findings of this study contribute to current understanding of the functions of miR-33b in osteosarcoma.
PMCID: PMC4278906  PMID: 25546234
18.  Choice of therapeutic strategies in intrathoracic anastomotic leak following esophagectomy 
The aim of this study was to analyze our experience with management of intrathoracic anastomotic leak after esophagectomy.
Clinical data from 33 patients who developed intrathoracic anastomotic leak were evaluated retrospectively. These patients were selected from 1867 patients undergoing resection carcinoma of the esophagus and reconstruction between January 2003 and December 2012.
Surgical intervention and the reformed “three-tube method” were applied in 13 and 20 patients, respectively. The overall incidence of intrathoracic anastomotic leakage was 1.8%. The median time interval from esophagectomy to diagnosis of leak was 9.7 days. Sixteen patients were confirmed as having leakage by oral contrast computed tomography (CT). Age and interval from surgery to diagnosis of leak were identified as statistically significant parameters between contained and uncontained groups. Moreover, patients with hypoalbuminemia had a longer time to leak closure than patients without hypoalbuminemia. Six patients died from intrathoracic anastomotic leak, with a mortality rate of 18.2%. There was no statistically significant difference in the time to leak closure between patients who underwent surgical exploration and those who received conservative treatment.
Intrathoracic anastomotic leak after esophagectomy was associated with significant mortality. Once intrathoracic anastomotic leakage following esophagectomy was diagnosed or highly suspected, individualized management strategies should be implemented according to the size of the leak, extent of the abscess, and status of the patient. In the majority of patients with anastomotic leak, we preferred the strategy of conservative treatment.
PMCID: PMC4320535  PMID: 25547979
19.  Superhydrophobic Photosensitizers. Mechanistic Studies of 1O2 Generation in the Plastron and Solid/Liquid Droplet Interface 
Journal of the American Chemical Society  2013;135(50):18990-18998.
We describe here a physical-organic study of the first triphasic superhydrophobic sensitizer for photooxidations in water droplets. Control of synthetic parameters enables the mechanistic study of “borderline” two- and three-phase superhydrophobic sensitizer surfaces where 1O2 is generated in compartments that are wetted, partially wetted, or remain dry in the plastron (i.e., air layer beneath the droplet). The superhydrophobic surface is synthesized by partially embedding silicon phthalocyanine (Pc) sensitizing particles to specific locations on polydimethylsiloxane (PDMS) posts printed in a square array (1 mm tall posts on 0.5 mm pitch). In the presence of red light and oxygen, singlet oxygen is formed on the superhydrophobic surface and reacts with 9,10-anthracene dipropionate dianion (1) within a freestanding water droplet to produce an endoperoxide in 54–72% yields. Control of the 1O2 chemistry was achieved by the synthesis of superhydrophobic surfaces enriched with Pc particles either at the PDMS end-tips or at PDMS post bases. Much of the 1O2 that reacts with anthracene 1 in the droplets was generated by the sensitizer “wetted” at the Pc particle/water droplet interface and gave the highest endoperoxide yields. About 20% of the 1O2 can be introduced into the droplet from the plastron. The results indicate that the superhydrophobic sensitizer surface offers a unique system to study 1O2 transfer routes where a balance of gas and liquid contributions of 1O2 is tunable within the same superhydrophobic surface.
PMCID: PMC3930608  PMID: 24295210
20.  Mmp1 and Mmp2 cooperatively induce Drosophila fat body cell dissociation with distinct roles 
Scientific Reports  2014;4:7535.
During Drosophila metamorphosis, the single-cell layer of fat body tissues gradually dissociates into individual cells. Via a fat body-specific RNAi screen in this study, we found that two matrix metalloproteinases (MMPs), Mmp1 and Mmp2, are both required for fat body cell dissociation. As revealed through a series of cellular, biochemical, molecular, and genetic experiments, Mmp1 preferentially cleaves DE-cadherin-mediated cell-cell junctions, while Mmp2 preferentially degrades basement membrane (BM) components and thus destroy cell-BM junctions, resulting in the complete dissociation of the entire fat body tissues into individual cells. Moreover, several genetic interaction experiments demonstrated that the roles of Mmp1 and Mmp2 in this developmental process are cooperative. In conclusion, Mmp1 and Mmp2 induce fat body cell dissociation during Drosophila metamorphosis in a cooperative yet distinct manner, a finding that sheds light on the general mechanisms by which MMPs regulate tissue remodeling in animals.
PMCID: PMC4269897  PMID: 25520167
21.  Epistatic Adaptive Evolution of Human Color Vision 
PLoS Genetics  2014;10(12):e1004884.
Establishing genotype-phenotype relationship is the key to understand the molecular mechanism of phenotypic adaptation. This initial step may be untangled by analyzing appropriate ancestral molecules, but it is a daunting task to recapitulate the evolution of non-additive (epistatic) interactions of amino acids and function of a protein separately. To adapt to the ultraviolet (UV)-free retinal environment, the short wavelength-sensitive (SWS1) visual pigment in human (human S1) switched from detecting UV to absorbing blue light during the last 90 million years. Mutagenesis experiments of the UV-sensitive pigment in the Boreoeutherian ancestor show that the blue-sensitivity was achieved by seven mutations. The experimental and quantum chemical analyses show that 4,008 of all 5,040 possible evolutionary trajectories are terminated prematurely by containing a dehydrated nonfunctional pigment. Phylogenetic analysis further suggests that human ancestors achieved the blue-sensitivity gradually and almost exclusively by epistasis. When the final stage of spectral tuning of human S1 was underway 45–30 million years ago, the middle and long wavelength-sensitive (MWS/LWS) pigments appeared and so-called trichromatic color vision was established by interprotein epistasis. The adaptive evolution of human S1 differs dramatically from orthologous pigments with a major mutational effect used in achieving blue-sensitivity in a fish and several mammalian species and in regaining UV vision in birds. These observations imply that the mechanisms of epistatic interactions must be understood by studying various orthologues in different species that have adapted to various ecological and physiological environments.
Author Summary
Mapping the genotype-phenotype relationship is necessary to understand how variable phenotypes have evolved in nature. The blue-sensitive visual pigment in human (human S1) evolved from the UV-sensitive pigment in the Boreoeutherian (or Boreotherian) ancestor (AncBoreotheria S1) by seven mutations. Mutagenesis experiments reveal that 4,008 (∼80%) of all 5,040 possible evolutionary trajectories connecting from AncBoreotheria S1 to human S1 are terminated prematurely. Quantum chemical analyses suggest that the premature termination of trajectories was caused by containing a dehydrated nonfunctional pigment. Phylogenetic analysis further suggests that the blue-sensitivity was achieved only gradually and almost exclusively by the seven non-additively interacting amino acids. During the period between 45 and 30 My ago, human S1 was in the final stage of developing its blue-sensitivity. This was the time when two red-sensitive pigments appeared by gene duplication and one of them became green-sensitive. Trichromatic color vision in the human lineage was fully developed by 30 My ago by interprotein epistasis among the three visual pigments. Manipulation of the genetically engineered ancestral molecule is the key to recapitulate the evolution of phenotypic adaptation and that of epistatic interaction separately.
PMCID: PMC4270479  PMID: 25522367
22.  Effect of temozolomide on livin and caspase-3 in U251 glioma stem cells 
The aim of the present study was to analyze the effect of temozolomide (TMZ) on the antiapoptotic gene livin and the associated gene caspase-3. Cancer stem cells were isolated from U251 glioblastoma cells using immunomagnetic beads. The glioma cells and glioma stem cells were transfected with livin or small hairpin RNA (shRNA) against livin using lentiviral vectors. Quantitative PCR, flow cytometry and a Cell Counting kit-8 assay were used to detect the expression of livin and caspase-3, analyze the cell cycle and investigate cell proliferation, respectively, following treatment with various concentrations of TMZ (0, 25, 50, 100, 200 and 400 μmol/l) for different periods of time (24, 48 and 72 h). The expression levels of livin and caspase-3 in the U251 stem cells were significantly higher than those in the U251 cells (P<0.01). At the same intervention time, the expression levels of livin decreased and those of caspase-3 increased as the concentration of TMZ increased (P<0.05). The expression levels of livin and caspase-3 in the U251 cells were lower than those in the U251 stem cells with the same intervention time and concentration of TMZ (P<0.05). The cell cycle was arrested in the G2/M phase in the U251 cells following TMZ intervention; the proportion of cells in the G2/M phase increased as the concentration of TMZ increased (P<0.05). The U251 stem cells were arrested in the S phase following treatment with TMZ; the proportion of cells in the S phase increased as the concentration of TMZ increased (P<0.05). In conclusion, the expression levels of livin and caspase-3 were effectively inhibited and increased, respectively, in all cell models following treatment with TMZ. TMZ is able to arrest the cell cycle and enhance cell apoptosis. U251 stem cells are less vulnerable than U251 cells to TMZ.
PMCID: PMC4316973  PMID: 25667622
glioma; stem cells; livin; caspase-3; cell cycle; temozolomide
23.  Flesh Quality Loss in Response to Dietary Isoleucine Deficiency and Excess in Fish: A Link to Impaired Nrf2-Dependent Antioxidant Defense in Muscle 
PLoS ONE  2014;9(12):e115129.
The present study explored the impact of dietary isoleucine (Ile) on fish growth and flesh quality and revealed a possible role of muscle antioxidant defense in flesh quality in relation to dietary Ile. Grass carp (weighing 256.8±3.5 g) were fed diets containing six graded levels of Ile (3.8, 6.6, 9.3, 12.5, 15.2 and 18.5 g/kg) for eight weeks. The results indicated that compared with Ile deficiency (3.8 g/kg diets) and excess (18.5 g/kg diets) groups, 9.3–15.2 g Ile/kg diet supplementations promoted fish growth and muscle fat deposition, whereas 6.6–15.2 g Ile/kg diets supplementation enhanced muscle nutrients (protein and total EAAs) deposition. Furthermore, muscle shear force, pH value, and hydroxyproline concentration were improved by 9.3–12.5, 9.3 and 9.3 g Ile/kg diet supplementations, respectively. However, muscle cooking loss, lactate content, and activities of cathepsin B and L were decreased by 6.6–15.2, 9.3–12.5, 9.3–12.5 and 9.3–15.2 g Ile/kg diet supplementations, respectively. Additionally, 6.6–15.2 and 6.6–12.5 g Ile/kg diet supplementations attenuated malondialdehyde and protein carbonyl contents, respectively. The activities of copper/zinc superoxide dismutase (Cu/Zn-SOD) and glutathione peroxidase (GPx), and glutathione content were enhanced by 6.6–9.3, 6.6–12.5 and 6.6–15.2 g Ile/kg diet supplementations, respectively. Moreover, the relative mRNA expressions of antioxidant enzymes, including Cu/Zn-SOD (6.6–12.5 g/kg diets) and GPx (12.5 g/kg diets), as well as antioxidant-related signaling molecules, including NF-E2-related factor 2 (Nrf2) (6.6–12.5 g/kg diets), target of rapamycin (6.6–12.5 g/kg diets), ribosomal S6 protein kinase 1 (9.3–12.5 g/kg diets) and casein kinase 2 (6.6–12.5 g/kg diets), were up-regulated when Ile diet supplementations were administered at these levels, respectively, whereas the relative mRNA expression of Kelch-like ECH-associated protein 1 was down-regulated with 9.3 g Ile/kg diet supplementations. Collectively, the present study indicated that optimum isoleucine improved flesh quality, partly due to the activation of antioxidant defense through the Nrf2 signaling pathway.
PMCID: PMC4267783  PMID: 25514235
24.  Red blood cell distribution width: a potential maker estimating disease activity of ankylosing spondylitis 
Ankylosing spondylitis (AS) is a autoimmune disease, early and accurate detection is vital for effective treatment. Very recently, a large of novel laboratory index were found to diagnose AS. However, the correlation between red blood cell distribution width (RDW) and AS has been poorly discussed in previous study. Then, our aim was to focus on the association between RDW and AS. AS patients without drugs treatments and healthy individuals were incorporated in our study. Laboratory parameters including RDW tests were conducted consecutively on the entire cohort of AS patients and healthy individuals. AS patients with increased RDW showed significant difference compared to healthy individuals (13.66±0.77 vs. 12.77±0.47, P<0.01), similarly, difference of AS patients and heathy individuals stratified by sex was almost the same as that in two groups. Some significantly positive correlation were observed between RDW and CRP, ESR, IgG, BASDAI score in patients with AS (r=0.356, P=0.018; r=0.481, P=0.001; r=0.385, P=0.010; r=0.586, P<0.01), almost identical results were showed when AS patients was stratified by gender. Finally, receiver operating characteristic (ROC) curves of RDW levels to identify AS patients exhibited a statistically significant level (area under the curve of 0.853; sensitivity of 72.7%, specificity of 81.4%). The results suggested that increased RDW was associated with AS and may be used as a potential marker estimating disease activity of AS.
PMCID: PMC4307480  PMID: 25664033
Ankylosing spondylitis; red blood cell distribution width; C-reactive protein; erythrocyte sedimentation rate; Bath AS disease activity index
25.  Effects of intrathecal anesthesia with different concentrations and doses on spinal cord, nerve roots and cerebrospinal fluid in dogs 
Objective: To investigate the effects of intrathecal anesthesia with bupivacaine, levobupivacaine and ropivacaine hydrochloride at different doses on the spinal cord, nerve roots and cerebrospinal fluid (CSF) in dogs. Methods: Forty-two mongrel dogs were randomly divided into normal saline group (C; 2 ml), 0.5% (B1) and 0.75% (B2) bupivacaine hydrochloride groups (2 ml), 0.5% (L1) and 0.75% (L2) levobupivacaine hydrochloride group (2 ml), 0.5% (R1) and 0.75% (R2) ropivacaine hydrochloride group (2 ml), and drugs were intrathecally injected. Results: The contents of Ca2+ and MDA and SOD activity of the spinal cord were comparable among groups (P > 0.05). In Groups B1, L1 and R1, the neuronal cytoplasm of spinal tissues was basically normal, the majority of mitochondria and endoplasmic reticulum had complete structure, and the lamellar structure of modulated fibers was nearly normal. In Groups B2, L2 and R2, a small amount of mitochondrial vacuolar degeneration was found in the neuronal cytoplasm of spinal cord, but their structures were basically normal; the neural tissues exhibited focal mild edema, and most of the lamellar structure of modulated fibers and Schwann cells were nearly normal except for loose structure in several fibers and cells. Conclusion: When compared with 0.75% anesthetics for local anesthesia, the early adverse effects on the ultrastructure of the spinal cord and nerve root reduce after focal anesthesia with 0.5% anesthetics.
PMCID: PMC4307493  PMID: 25664046
Amides; intrathecal anesthesia; spinal cord; nerve root; ultrastructure; free radical; Ca2+

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