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1.  VEGF, not VEGFR2, is associated with the angiogenesis effect of mini-TyrRS/mini-TrpRS in human umbilical vein endothelial cells in hypoxia 
Cytotechnology  2013;66(4):655-665.
The purpose of this study was to determine the relationship between VEGF and mini-TyrRS/mini-TrpRS in angiogenesis in hypoxic culture and to begin to comprehend their mechanism in angiogenesis. We designed a VEGF gene silencing assay by using lentivirus vectors, and then western blotting was used to determine the protein expression of VEGF, VEGFR2 and pVEGFR2 in three groups in hypoxic culture at 3, 6, 12, or 24 h: (1) untransfected human umbilical vein endothelial cells (HUVECs) (Control); (2) pGCSIL-GFP lentivirus vector-transduced HUVECs (Mock); and (3) pGCSIL-shVEGF lentivirus vector-transduced HUVECs (Experimental). We also detected the effects of mini-TyrRS/mini-TrpRS peptides on HUVEC proliferation, migration and tube formation after lentivirus vector transfection and VEGFR2 antibody injection. The results indicated that expression of the mini-TyrRS protein was increased, whereas that of mini-TrpRS was specifically decreased in hypoxic culture both in control and mock groups. However, this trend in protein levels of mini-TyrRS and mini-TrpRS was lost in the experimental group after transduction with the pGCSIL-shVEGF lentivirus vector. The protein expression of VEGF was increased in hypoxic culture both in control and mock groups. After transduction with the pGCSIL-shVEGF lentivirus vector, the protein level of VEGF was noticeably decreased in the experimental group; however, for VEGFR2, the results showed no significant difference in VEGFR2 protein expression in any of the groups. For pVEGFR2, we found a distinct trend from that seen with VEGF. The protein expression of pVEGFR2 was sharply increased in hypoxic culture in the three groups. The addition of mini-TyrRS significantly promoted proliferation, migration and tube formation of HUVECs, while mini-TrpRS inhibited these processes in both control and mock groups in hypoxic culture. However, these effects disappeared after transduction with the pGCSIL-shVEGF lentivirus vector in the experimental group, but no significant difference was observed after VEGFR2 antibody injection. The protein expression of VEGF is similar to that of mini-TyrRS in hypoxic culture and plays an important role in the mini-TyrRS/mini-TrpRS-stimulated proliferation, migration and tube formation of HUVECs in hypoxia. These results also suggest that the change in mini-TyrRS and mini-TrpRS expression in hypoxic culture is not related to VEGFR2 and that some other possible mechanisms, are involved in the phosphorylation of VEGFR2.
doi:10.1007/s10616-013-9619-6
PMCID: PMC4082773  PMID: 23896703
Mini-TyrRS; Mini-TrpRS; Angiogenesis; VEGF; VEGFR2; Hypoxia; HUVEC
2.  Somatostatin Improved B Cells Mature in Macaques during Intestinal Ischemia-Reperfusion 
PLoS ONE  2015;10(7):e0133692.
Aims
Intestinal ischemia-reperfusion has been taken as an important pathophysiological process for multiple organ dysfunctions in critical patients. Recent studies reported that dual expression programs of the B cells receptors and Toll-like receptors on B-lymphocytes permit these ubiquitous cells to integrate both adaptive and innate immune functions. Our previous studies found that somatostatin inhibited the intestinal inflammatory injury after ischemia-reperfusion in macaques. However, the changes of B cells and the effects of somatostatin on B cells after intestinal ischemia-reperfusion were unclear.
Methods
15 macaques were divided into control, intestinal ischemia-reperfusion and somatostatin pretreatment groups. Immunohistochemistry was performed to identify the distributions of adaptive and innate immunity markers in the iliac mucosa. Hmy2.cir B lymphoblastoid cell line was cultured in vitro study. Enzyme-linked immunosorbent assay was used to measure IgM, IL-6 and SIgA, and the expressions of B cells transcription factors, PAX-5 and BLIMP-1, were detected by Western blotting.
Results
B2 lymphocytes in normal Peyer’s patches were presented the phenotype of PAX-5+CD20+CD5-. Ischemia-reperfusion increased the numbers and sizes of Peyer’s patches but with PAX-5+CD20-CD5- B cells, an unmatured set of B cells. Somatostatin partly kept the phenotype of mature B cells during ischemia-reperfusion. The innate immunity of B cells was inhibited whereas the adaptive immunity was increased in the intestinal mucosa in the somatostatin group, compared to the ischemia-reperfusion group. In vitro, somatostatin significantly inhibited IL-6 and promoted IgM by increasing the expression of both PAX-5 and BLIMP-1 in the proinflammatory condition.
Conclusion
Intestinal ischemia-reperfusion resulted in the proliferation of unmatured B cells which were involved in the augmentation of innate immunity. Somatostatin, with a bi-directional regulation function on innate as well as adaptive immunity of B cells, greatly improved B cells mature in macaques during ischemia-reperfusion. Preventive supplements of somatostatin may greatly limit intestinal injury and bacterial translocation during ischemia-reperfusion.
doi:10.1371/journal.pone.0133692
PMCID: PMC4519283  PMID: 26222793
3.  The reliability and validity of a Chinese-version Short Health Anxiety Inventory: an investigation of university students 
Background
The Short Health Anxiety Inventory (SHAI) is widely used in English-speaking populations, with good reliability and validity. For further research needs in the Chinese population, it was translated into a Chinese version (CSHAI). Furthermore, the reliability, validity, and cutoff score were examined in a nonclinical population in the People’s Republic of China.
Methods
Three hundred and sixteen undergraduates were evaluated by a set of questionnaires including CSHAI, Zung Self-Rating Anxiety Scale (SAS), Zung Self-Rating Depression Scale (SDS), and the State-Trait Anxiety Inventory (STAI). Fifty-eight students completed CSHAI again after 30 days.
Results
The two-factor model had satisfactory fit indices. The correlation coefficients between each item with the CSHAI total and each subscale were between 0.386 and 0.779. The Cronbach’s alpha coefficients of CSHAI total and its subscales were 0.742, 0.743, and 0.788, respectively, and the split-half coefficients were 0.757, 0.788, and 0.912. The test–retest correlation coefficients were, respectively, 0.598 (P<0.001), 0.539 (P<0.001), and 0.691 (P<0.001). Convergent validities were respectively 0.389–0.453, 0.389–0.410, and 0.250–0.401, and discriminant validities were −5.689 (P<0.001), −5.614 (P<0.001), and −3.709 (P<0.001). The cutoff score was 15.
Conclusion
CSHAI showed good factor structure, reliability, convergent validity, and discriminant validity, and 15 was determined to be the appropriate cutoff score for screening health anxiety.
doi:10.2147/NDT.S83501
PMCID: PMC4509540  PMID: 26213472
health anxiety; confirmatory factor analysis; cutoff score
4.  Itraconazole suppresses the growth of glioblastoma through induction of autophagy 
Autophagy  2014;10(7):1241-1255.
Glioblastoma is one of the most aggressive human cancers with poor prognosis, and therefore a critical need exists for novel therapeutic strategies for management of glioblastoma patients. Itraconazole, a traditional antifungal drug, has been identified as a novel potential anticancer agent due to its inhibitory effects on cell proliferation and tumor angiogenesis; however, the molecular mechanisms involved are still unclear. Here, we show that itraconazole inhibits the proliferation of glioblastoma cells both in vitro and in vivo. Notably, we demonstrate that treatment with itraconazole induces autophagic progression in glioblastoma cells, while blockage of autophagy markedly reverses the antiproliferative activities of itraconazole, suggesting an antitumor effect of autophagy in response to itraconazole treatment. Functional studies revealed that itraconazole retarded the trafficking of cholesterol from late endosomes and lysosomes to the plasma membrane by reducing the levels of SCP2, resulting in repression of AKT1-MTOR signaling, induction of autophagy, and finally inhibition of cell proliferation. Together, our studies provide new insights into the molecular mechanisms regarding the antitumor activities of itraconazole, and may further assist both the pharmacological investigation and rational use of itraconazole in potential clinical applications.
doi:10.4161/auto.28912
PMCID: PMC4203550  PMID: 24905460
itraconazole; antiproliferative; autophagy; BECN1-PtdIns3K complex; cholesterol trafficking
5.  Investigation of Ground-Level Ozone and High-Pollution Episodes in a Megacity of Eastern China 
PLoS ONE  2015;10(6):e0131878.
Differential Optical Absorption Spectroscopy (DOAS) was used for the long-term observation of ground-level ozone (O3) from March 2010 to March 2013 over Shanghai, China. The 1-hour average concentration of O3 was 27.2 ± 17.0 ppbv. O3 level increased during spring, reached the peak in late spring and early summer, and then decreased in autumn and finally dropped to the bottom in winter. The highest monthly average O3 concentration in June (41.1 ppbv) was nearly three times as high as the lowest level recorded in December (15.2 ppbv). In terms of pollution episodes, 56 hourly samples (on 14 separate days) in 2010 exceeded the 1-hour ozone limit of 200 μg/m3 specified by the Grade II of the Chinese Ambient Air Quality Standards (CAAQS, revised GB 3095-2012). Utilizing the Hybrid Single Particle Lagrangian Integrated Trajectory (HYSPLIT) model, the primary contribution to high ozone days (HODs) was identified as the regional transportation of volatile organic compounds (VOC) and high concentrations of O3 from the chemical industrial zone in the Jinshan district of Shanghai. HODs showed higher concentrations of HONO and NO2 than non-episode conditions, implying that HONO at high concentration during HODs was capable of increasing the O3 concentration. The photolysis rate of HONO was estimated, suggesting that the larger number of OH radicals resulting from high concentrations of HONO have a considerable impact on ozone concentrations.
doi:10.1371/journal.pone.0131878
PMCID: PMC4486460  PMID: 26121146
6.  PsANT, the adenine nucleotide translocase of Puccinia striiformis, promotes cell death and fungal growth 
Scientific Reports  2015;5:11241.
Adenine nucleotide translocase (ANT) is a constitutive mitochondrial component that is involved in ADP/ATP exchange and mitochondrion-mediated apoptosis in yeast and mammals. However, little is known about the function of ANT in pathogenic fungi. In this study, we identified an ANT gene of Puccinia striiformis f. sp. tritici (Pst), designated PsANT. The PsANT protein contains three typical conserved mitochondrion-carrier-protein (mito-carr) domains and shares more than 70% identity with its orthologs from other fungi, suggesting that ANT is conserved in fungi. Immuno-cytochemical localization confirmed the mitochondrial localization of PsANT in normal Pst hyphal cells or collapsed cells. Over-expression of PsANT indicated that PsANT promotes cell death in tobacco, wheat and fission yeast cells. Further study showed that the three mito-carr domains are all needed to induce cell death. qRT-PCR analyses revealed an in-planta induced expression of PsANT during infection. Knockdown of PsANT using a host-induced gene silencing system (HIGS) attenuated the growth and development of virulent Pst at the early infection stage but not enough to alter its pathogenicity. These results provide new insight into the function of PsANT in fungal cell death and growth and might be useful in the search for and design of novel disease control strategies.
doi:10.1038/srep11241
PMCID: PMC4462048  PMID: 26058921
7.  miR-135b Promotes Cancer Progression by Targeting Transforming Growth Factor Beta Receptor II (TGFBR2) in Colorectal Cancer 
PLoS ONE  2015;10(6):e0130194.
The transforming growth factor beta (TGF-β) signaling pathway is a tumor-suppressor pathway that is commonly inactivated in colorectal cancer (CRC). The inactivation of TGFBR2 is the most common genetic event affecting the TGF-β signaling pathway. However, the mechanism by which cancer cells downregulate TGFBR2 is unclear. In this study, we found that the TGFBR2 protein levels were consistently upregulated in CRC tissues, whereas its mRNA levels varied in these tissues, suggesting that a post-transcriptional mechanism is involved in the regulation of TGFBR2. Because microRNAs (miRNAs) are powerful post-transcriptional regulators of gene expression, we performed bioinformatic analyses to search for miRNAs that potentially target TGFBR2. We identified the specific targeting site of miR-135b in the 3’-untranslated region (3’-UTR) of TGFBR2. We further identified an inverse correlation between the levels of miR-135b and TGFBR2 protein, but not mRNA, in CRC tissue samples. By overexpressing or silencing miR-135b in CRC cells, we experimentally validated that miR-135b directly binds to the 3’-UTR of the TGFBR2 transcript and regulates TGFBR2 expression. Furthermore, the biological consequences of the targeting of TGFBR2 by miR-135b were examined using in vitro cell proliferation and apoptosis assays. We demonstrated that miR-135b exerted a tumor-promoting effect by inducing the proliferation and inhibiting the apoptosis of CRC cells via the negative regulation of TGFBR2 expression. Taken together, our findings provide the first evidence supporting the role of miR-135b as an oncogene in CRC via the inhibition of TGFBR2 translation.
doi:10.1371/journal.pone.0130194
PMCID: PMC4462589  PMID: 26061281
8.  Review on the Applications and Molecular Mechanisms of Xihuang Pill in Tumor Treatment 
Xihuang pill (XH) is a complementary and alternative medicine that has been used in traditional Chinese medicine (TCM) for the treatment of tumors since the 18th century. XH has clinical effects on non-Hodgkin lymphoma, breast cancer, gastric cancer, liver cancer, and bone metastasis. XH can also inhibit the growth of tumor cells and cancer stem cells, prevent tumor invasion and angiogenesis, and regulate the tumor microenvironment. XH is composed of Ru Xiang (olibanum), Mo Yao (Commiphora myrrha), She Xiang (Moschus), and Niu Huang (Calculus bovis). Some of the compounds found in these ingredients exert multiple antitumor effects and may synergize with the other ingredients. We aimed to summarize the clinical applications and molecular mechanisms of XH and its chemical composition. This review will provide potential new strategies and alternative perspectives for tumor treatments and basic research into complementary and alternative medicine.
doi:10.1155/2015/854307
PMCID: PMC4479127  PMID: 26170886
9.  DNA Marker Transmission and Linkage Analysis in Populations Derived from a Sugarcane (Saccharum spp.) x Erianthus arundinaceus Hybrid 
PLoS ONE  2015;10(6):e0128865.
Introgression of Erianthus arundinaceus has been the focus of several sugarcane breeding programs in the world, because the species has desirable traits such as high biomass production, vigour, ratooning ability and good resistance to environmental stresses and disease. In this study four genetic maps were constructed for two intergeneric populations. The first population (BC1) was generated from a cross between an Erianthus/Saccharum hybrid YC96-40 and a commercial sugarcane variety CP84-1198. The second population (BC2) was generated from a cross between YCE01-116, a progeny of the BC1 cross and NJ57-416, a commercial sugarcane cultivar. Markers across both populations were generated using 35 AFLP and 23 SSR primer pairs. A total of 756 and 728 polymorphic markers were scored in the BC1 and BC2 populations, respectively. In the BC1 population, a higher proportion of markers was derived from the Erianthus ancestor than those from the Saccharum ancestor Badila. In the BC2 population, both the number and proportion of markers derived from Erianthus were approximately half of those in the BC1 population. Linkage analysis led to the construction of 38, 57, 36 and 47 linkage groups (LGs) for YC96-40, CP84-1198, YCE01-116, and NJ57-416, encompassing 116, 174, 97 and 159 markers (including single dose, double dose and bi-parental markers), respectively. These LGs could be further placed into four, five, five and six homology groups (HGs), respectively, based on information from multi-allelic SSR markers and repulsion phase linkages detected between LGs. Analysis of repulsion phase linkage indicated that Erianthus behaved like a true autopolyploid.
doi:10.1371/journal.pone.0128865
PMCID: PMC4459986  PMID: 26053338
10.  Female reproductive factors, menopausal hormone use, and Parkinson’s disease 
Objective
To examine the associations of reproductive factors and exogenous hormone use with risk of Parkinson’s disease (PD) among postmenopausal women.
Methods
The study comprised 119,166 postmenopausal women ages 50–71 years in the NIH-AARP Diet and Health Study, who completed a baseline questionnaire in 1995–1996 and a follow-up survey in 2004–2006. A total of 410 self-reported PD diagnoses were identified between 1995 and 2006. Multivariate odds ratios (OR) and 95% confidence intervals (CI) were derived from logistic regression models.
Results
PD risk was not significantly associated with female reproductive factors including age at menarche, age at first live birth, parity, and age at menopause. For example, compared with women with natural menopause at ages 50–54 years, the ORs were 1.18, (95% CI 0.78–1.79) for women with natural menopause at ages <45, 1.19 (0.88–1.61) for ages 45–49, and 1.33 (0.91–1.93) for ages 55 or later. We found that oral contraceptive use for ≥10 years (vs. never use) was associated with lower PD risk (OR=0.59; 0.38–0.92) but shorter use showed no association. Use of menopausal hormone therapy showed inconsistent results. Compared with non-hormone users at baseline, current hormone users of <5 years showed a higher risk of PD (OR=1.52; 1.11–2.08). However, no associations were observed for past hormone users or current users of ≥5 years.
Conclusions
Overall, this large prospective study provides little support for an association between female reproductive factors and PD risk. Our findings on long-term oral contraceptive use and current hormone therapy warrant further investigations.
doi:10.1002/mds.25771
PMCID: PMC4057969  PMID: 24352877
Reproductive factors; Parkinson’s disease; cohort studies; menopausal hormone therapy
11.  Comparative Assessment of Phenolic Content and in Vitro Antioxidant Capacity in the Pulp and Peel of Mango Cultivars 
Mango (Mangifera indica L.), also called “the king of fruits”, is one of the most popular fruits in tropical regions. Pulp and peel samples of mango cultivars were analyzed to estimate total phenolic, total flavonoid and total anthocyanin contents. Phenolic acids, hydrophilic peroxyl radical scavenging capacity (hydro-PSC) and oxygen radical scavenging capacity (ORAC) in vitro were also determined. Total phenolics and flavonoid contents were found maximum in the peel of Xiao Tainang and Da Tainang cultivars, respectively, whereas Xiao Tainang also exhibited significant antioxidant capacity. Noteworthy, concentrations of gallic acid, protocatechuic acid, ferulic acid, chlorogenic acid and caffeic acids at 79.15, 64.33, 33.75, 27.19 and 13.62 mg/100 g fresh weight (FW) were quantified for Da Tainang, Xiao Tainang and of Jidan cultivars, respectively. Comparatively, a higher level of phenolics and significant antioxidant capacity in mango peel indicated that it might be useful as a functional food and value-added ingredient to promote human health.
doi:10.3390/ijms160613507
PMCID: PMC4490506  PMID: 26075869
mango; phenolics; flavonoids; anthocyanins; phenolic acids; ORAC; hydro-PSC
12.  Witnessing magnetic twist with high-resolution observation from the 1.6-m New Solar Telescope 
Nature Communications  2015;6:7008.
Magnetic flux ropes are highly twisted, current-carrying magnetic fields. They are crucial for the instability of plasma involved in solar eruptions, which may lead to adverse space weather effects. Here we present observations of a flaring using the highest resolution chromospheric images from the 1.6-m New Solar Telescope at Big Bear Solar Observatory, supplemented by a magnetic field extrapolation model. A set of loops initially appear to peel off from an overall inverse S-shaped flux bundle, and then develop into a multi-stranded twisted flux rope, producing a two-ribbon flare. We show evidence that the flux rope is embedded in sheared arcades and becomes unstable following the enhancement of its twists. The subsequent motion of the flux rope is confined due to the strong strapping effect of the overlying field. These results provide a first opportunity to witness the detailed structure and evolution of flux ropes in the low solar atmosphere.
Understanding the behaviour of magnetic flux ropes in the Sun is crucial for explaining solar phenomena such as flares and space weather. Exploiting the high resolution available in the 1.6 m New Solar Telescope, Wang et al. capture the evolution of a flaring twisted flux rope in the low solar corona.
doi:10.1038/ncomms8008
PMCID: PMC4421823  PMID: 25919706
13.  Sox2 function as a negative regulator to control HAMP expression 
Biological Research  2015;48(1):23.
Background
Hepcidin, encoding by HAMP gene, is the pivotal regulator of iron metabolism, controlling the systemic absorption and transportation of irons from intracellular stores. Abnormal levels of HAMP expression alter plasma iron parameters and lead to iron metabolism disorders. Therefore, it is an important goal to understand the mechanisms controlling HAMP gene expression.
Results
Overexpression of Sox2 decrease basal expression of HAMP or induced by IL-6 or BMP-2, whereas, knockdown of Sox2 can increase HAMP expression, furthermore, two potential Sox2-binding sites were identified within the human HAMP promoter. Indeed, luciferase experiments demonstrated that deletion of any Sox2-binding site impaired the negative regulation of Sox2 on HAMP promoter transcriptional activity in basal conditions. ChIP experiments showed that Sox2 could directly bind to these sites. Finally, we verified the role of Sox2 to negatively regulate HAMP expression in human primary hepatocytes.
Conclusion
We found that Sox2 as a novel factor to bind with HAMP promoter to negatively regulate HAMP expression, which may be further implicated as a therapeutic option for the amelioration of HAMP-overexpression-related diseases, including iron deficiency anemia.
Electronic supplementary material
The online version of this article (doi:10.1186/s40659-015-0013-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s40659-015-0013-z
PMCID: PMC4440282  PMID: 25943891
Sox2; Gene expression; Hepatocyte; HAMP; Iron metabolism
14.  Placental mosaicism for Trisomy 13: a challenge in providing the cell-free fetal DNA testing 
Purpose
We investigated the disagreement between the positive cell-free fetal DNA test for trisomy 13 and the standard cytogenetic diagnosis of one case.
Methods
Cell-free fetal DNA testing was performed by massively parallel sequencing. We used conventional cytogenetic analysis to confirm the commercial cell-free fetal DNA testing. Additionally, postnatal fluorescent in situ hybridization (FISH) testing was performed on placental tissues.
Results
The cell-free fetal DNA testing result was positive for trisomy 13. G-banded analysis of amniotic fluid was normal, 46, XY. FISH testing of tissues from four quadrants of the placenta demonstrated mosaicism for trisomy 13.
Conclusions
A positive cell-free fetal DNA testing result may not be representative of the fetal karyotype because of placental mosaicism. Cytogenetic analysis should be performed when abnormal cell-free fetal DNA test results are obtained.
doi:10.1007/s10815-014-0182-7
PMCID: PMC4016375  PMID: 24497298
Noninvasive prenatal diagnosis; Positive cell-free fetal DNA testing; Trisomy 13; Placental mosaicism
15.  Eukaryotic Elongation Factor 2 Kinase Activity Is Controlled by Multiple Inputs from Oncogenic Signaling 
Molecular and Cellular Biology  2014;34(22):4088-4103.
Eukaryotic elongation factor 2 kinase (eEF2K), an atypical calmodulin-dependent protein kinase, phosphorylates and inhibits eEF2, slowing down translation elongation. eEF2K contains an N-terminal catalytic domain, a C-terminal α-helical region and a linker containing several regulatory phosphorylation sites. eEF2K is expressed at high levels in certain cancers, where it may act to help cell survival, e.g., during nutrient starvation. However, it is a negative regulator of protein synthesis and thus cell growth, suggesting that cancer cells may possess mechanisms to inhibit eEF2K under good growth conditions, to allow protein synthesis to proceed. We show here that the mTORC1 pathway and the oncogenic Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) pathway cooperate to restrict eEF2K activity. We identify multiple sites in eEF2K whose phosphorylation is regulated by mTORC1 and/or ERK, including new ones in the linker region. We demonstrate that certain sites are phosphorylated directly by mTOR or ERK. Our data reveal that glycogen synthase kinase 3 signaling also regulates eEF2 phosphorylation. In addition, we show that phosphorylation sites remote from the N-terminal calmodulin-binding motif regulate the phosphorylation of N-terminal sites that control CaM binding. Mutations in the former sites, which occur in cancer cells, cause the activation of eEF2K. eEF2K is thus regulated by a network of oncogenic signaling pathways.
doi:10.1128/MCB.01035-14
PMCID: PMC4248706  PMID: 25182533
16.  The miRNA Transcriptome Directly Reflects the Physiological and Biochemical Differences between Red, White, and Intermediate Muscle Fiber Types 
MicroRNAs (miRNAs) are small non-coding RNAs that can regulate their target genes at the post-transcriptional level. Skeletal muscle comprises different fiber types that can be broadly classified as red, intermediate, and white. Recently, a set of miRNAs was found expressed in a fiber type-specific manner in red and white fiber types. However, an in-depth analysis of the miRNA transcriptome differences between all three fiber types has not been undertaken. Herein, we collected 15 porcine skeletal muscles from different anatomical locations, which were then clearly divided into red, white, and intermediate fiber type based on the ratios of myosin heavy chain isoforms. We further illustrated that three muscles, which typically represented each muscle fiber type (i.e., red: peroneal longus (PL), intermediate: psoas major muscle (PMM), white: longissimus dorsi muscle (LDM)), have distinct metabolic patterns of mitochondrial and glycolytic enzyme levels. Furthermore, we constructed small RNA libraries for PL, PMM, and LDM using a deep sequencing approach. Results showed that the differentially expressed miRNAs were mainly enriched in PL and played a vital role in myogenesis and energy metabolism. Overall, this comprehensive analysis will contribute to a better understanding of the miRNA regulatory mechanism that achieves the phenotypic diversity of skeletal muscles.
doi:10.3390/ijms16059635
PMCID: PMC4463610  PMID: 25938964
miRNA; fiber type; pig; myogenesis; energy metabolism
17.  A cortical astrocyte subpopulation inhibits axon growth in vitro and in vivo 
Molecular Medicine Reports  2015;12(2):2598-2606.
Astrocytes are the most heterogeneous and predominant glial cell type in the central nervous system. However, the functional significance of this heterogeneity remains to be elucidated. Following injury, damaged astrocytes inhibit axonal regeneration in vivo and in vitro. Cultured primary astrocytes are commonly considered good supportive substrates for neuron attachment and axon regeneration. However, it is not known whether different populations of cells in the heterogeneous astrocyte culture affect neuron behavior in the same way. In the present study, the effect of astrocyte heterogeneity on neuronal attachment and neurite outgrowth was examined using an in vitro and in vivo co-culture system. In vitro, neonatal cortical astrocytes were co-cultured with purified dorsal root ganglia (DRG) neurons and astrocyte growth morphology, neuron attachment and neurite growth were evaluated. The results demonstrated that the heterogeneous astrocyte cells showed two different types of growth pattern, typical and atypical. Typical astrocytes were supportive to neuron attachment and neurite growth, which was consistent with previous studies, whereas atypical astrocytes inhibited neuron attachment and neurite growth. These inhibitory astrocytes exhibited a special growth pattern with various shapes and sizes, a high cell density, few oligodendrocytes on the top layer and occupied a smaller growth area compared with typical astrocytes. Neurites extended freely on typical supportive astrocyte populations, however, moved away when they reached atypical astrocyte growth pattern. Neurons growing on the atypical astrocyte pattern demonstrated minimal neurite outgrowth and these neurites had a dystrophic appearance, however, neuronal survival was unaffected. Immunocytochemistry studies demonstrated that these atypical inhibitory astrocytes were glial fibrillary acidic protein (GFAP) positive cells. The existence of inhibitory astrocyte subpopulations in normal astrocytes reflects the complexity of the function of astrocyte populations. In vivo, DRG neurons in grey matter did not show neurite growth, while DRG neurons survived and showed robust axon outgrowth along the corpus callosum. In conclusion, further studies on this new type of inhibitory astrocyte subpopulation may deepen our understanding of the complex biology of astrocytes.
doi:10.3892/mmr.2015.3702
PMCID: PMC4464481  PMID: 25936767
astrocyte; heterogeneous; axon growth; dorsal root ganglia neurons
18.  Remodeling of Kv1.5 Channel in Right Atria from Han Chinese Patients with Atrial Fibrillation 
Background
The incidence of atrial fibrillation (AF) in rheumatic heart diseases (RHD) is very high and increases with age. Occurrence and maintenance of AF are very complicated process accompanied by many different mechanisms. Ion-channel remodeling, including the voltage-gated potassium channel Kv1.5, plays an important role in the pathophysiology of AF. However, the changes of Kv1.5 channel expression in Han Chinese patients with RHD and AF remain poorly understood. The aim of the present study was to investigate whether the Kv1.5 channels of the right atria may be altered with RHD, age, and sex to contribute to AF.
Material/Methods
Right atrial appendages were obtained from 20 patients with normal cardiac functions who had undergone surgery, and 26 patients with AF. Subjects were picked from 4 groups: adult and aged patients in normal sinus rhythm (SR) and AF. Patients were divided into non-RHD and RHD groups or men and women groups in normal SR and AF, respectively. The expression of Kv1.5 protein and messenger RNA (mRNA) were measured using Western blotting and polymerase chain reaction (PCR) method, respectively.
Results
Compared with the SR group, the expression of Kv1.5 protein decreased significantly in the AF group. However, neither Kv1.5 protein nor KCNA5 mRNA had significant differences in adult and aged groups, non-RHD and RHD group, and men and women group of AF.
Conclusions
The expression of Kv1.5 channel protein changes with AF but not with age, RHD, and sex in AF.
doi:10.12659/MSM.893533
PMCID: PMC4424910  PMID: 25918274
Aging; Atrial Fibrillation; Kv1.5 Potassium Channel; Rheumatic Heart Disease; Sex
19.  Reduced ubiquitin-specific protease 9X expression induced by RNA interference inhibits the bioactivity of hepatocellular carcinoma cells 
Oncology Letters  2015;10(1):268-272.
Ubiquitin-specific protease 9X (USP9X) is crucial in many tumor types, but not in hepatocellular carcinoma (HCC). The current study aimed to examine the effects of RNA interference on USP9X expression, and subsequently on the bioactivity of HCC SMMC7721 and HepG2 cells. The protein expression of USP9X in SMMC7721, HepG2 and normal human liver cell line L02 at the cellular level was determined by western blot analysis; USP9X was knocked down by small interfering RNA (siRNA) in HCC SMMC7721 and HepG2 cells. In vitro cell viability was assessed by MTT assay, apoptosis was determined by flow cytometry (FCM) and cell migration was evaluated by Transwell assays. The protein expression of USP9X in SMMC7721 and HepG2 were both significantly higher than that in L02 (P<0.01). The results of western blot demonstrated that the USP9X-siRNA could efficiently inhibit USP9X expression when compared with that of the negative control (NC) group (P<0.01) and MTT assay demonstrated that cell proliferation in USP9X-blocked cells was significantly reduced when compared with that of the NC group (P<0.01). The results of FCM revealed that apoptosis was significantly increased in USP9X-blocked cells when compared with that of the NC group (P<0.01). The results of transwell assay showed that cell migration was significantly inhibited in USP9X-blocked cells when compared with that of the NC group (P<0.01). These results show that expression of USP9X is upregulated in hepatoma cells SMMC7721 and HepG2, and that downregulating USP9X by siRNA may induce cell apoptosis, inhibit cell growth and cell migration in the HCC SMMC7721 and HepG2 cell lines. USP9X may therefore be a potential target for HCC treatment and early detection.
doi:10.3892/ol.2015.3152
PMCID: PMC4487130  PMID: 26171012
apoptosis; growth; hepatocellular carcinoma; migration; RNA interference; ubiquitin-specific protease 9X
20.  Effect of the beta-3 adrenergic receptor Trp64Arg and uncoupling protein 1–3826 A > G genotypes on lipid and apolipoprotein levels in overweight/obese and non-obese Chinese subjects 
Background
The beta-3 adrenergic receptor (β3-AR) Trp64Arg and uncoupling protein 1 (UCP1) -3826 A > G polymorphisms have been reported to be associated with obesity and/or lipid metabolism in some populations. This study examined the possible association of the β3-AR and UCP1 polymorphisms with overweight/obesity or lipid variation in a Southwest Chinese population.
Methods
A total of 418 Han Chinese (249 overweight/obese and 169 healthy control subjects) in the Chengdu area were studied using PCR-RFLP analysis. Total serum cholesterol (TC) and triglycerides (TGs) were measured using an enzymatic method. High density lipoprotein cholesterol (HDL-C) was determined after sodium phosphotungstate/magnesium chloride precipitation of low-density lipoproteins by polyvinyl sulfate. Serum apolipoproteins were quantified by radial immunodiffusion.
Results
The genotype and allele frequencies of the β3-AR Trp64Arg and UCP1 -3826 A > G polymorphisms in overweight/obese subjects exhibited no significant differences compared to the controls. However, subjects carrying the β3-AR TrpTrp genotype and UCP1 AG genotype had higher TG levels than those carrying the Arg allele and AA genotype, respectively (P < 0.05), while controls carrying the β3-AR Arg allele had significantly higher TC and apo AII concentrations than those carrying the TrpTrp genotype (P < 0.05). Additionally, subjects carrying the UCP1 AG genotype exhibited elevated apo C-II and apo C-III levels compared to those carrying the AA genotype (P < 0.05). We were unable to find an association of the UCP1 and β3-AR polymorphisms with low HDL-cholesterolemia in the overweight/obese subjects.
Conclusions
The present study provides evidence that the β3-AR Trp64Arg and UCP1 -3826 A > G polymorphisms are associated with TG levels in overweight/obese Chinese subjects and that the two polymorphisms are also associated with certain lipid and apolipoprotein variations, depending on BMI. However, these polymorphisms are not associated with overweight/obesity or low HDL-cholesterolemia in a Chinese population from the Chengdu area.
doi:10.1186/s12944-015-0029-y
PMCID: PMC4410578  PMID: 25928572
Overweight/obese; \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \beta $$\end{document}β3-AR; UCP1; Gene polymorphism; PCR-RFLP
21.  Exposure Medium: Key in Identifying Free Ag+ as the Exclusive Species of Silver Nanoparticles with Acute Toxicity to Daphnia magna 
Scientific Reports  2015;5:9674.
It is still not very clear what roles the various Ag species play in the toxicity of silver nanoparticles (AgNPs). In this study, we found that traditional exposure media result in uncontrollable but consistent physicochemical transformation of AgNPs, causing artifacts in determination of median lethal concentration (LC50) and hindering the identification of Ag species responsible for the acute toxicity of AgNPs to Daphnia magna. This obstacle was overcome by using 8 h exposure in 0.1 mmol L−1 NaNO3 medium, in which we measured the 8-h LC50 of seven AgNPs with different sizes and coatings, and determined the concentrations of various Ag species. The LC50 as free Ag+ of the seven AgNPs (0.37–0.44 μg L−1) agreed very well with that of AgNO3 (0.40 μg L−1), and showed the lowest value compared to that as total Ag, total Ag+, and dissolved Ag, demonstrating free Ag+ is exclusively responsible for the acute toxicity of AgNPs to D. magna, while other Ag species in AgNPs have no contribution to the acute toxicity. Our results demonstrated the great importance of developing appropriate exposure media for evaluating risk of nanomaterials.
doi:10.1038/srep09674
PMCID: PMC4392358  PMID: 25858866
22.  Multiplexed aberration measurement for deep tissue imaging in vivo 
Nature methods  2014;11(10):1037-1040.
We describe a multiplexed aberration measurement method that modulates the intensity or phase of light rays at multiple pupil segments in parallel to determine their phase gradients. Applicable to fluorescent-protein-labeled structures of arbitrary complexity, it allows us to obtain diffraction-limited resolution in various samples in vivo. For the strongly scattering mouse brain, a single aberration correction improves structural and functional imaging of fine neuronal processes over a large imaging volume.
doi:10.1038/nmeth.3068
PMCID: PMC4180771  PMID: 25128976
23.  Mesenchymal Stem Cells from Patients with Rheumatoid Arthritis Display Impaired Function in Inhibiting Th17 Cells 
Journal of Immunology Research  2015;2015:284215.
Mesenchymal stem cells (MSCs) possess multipotent and immunomodulatory properties and are suggested to be involved in the pathogenesis of immune-related diseases. This study explored the function of bone marrow MSCs from rheumatoid arthritis (RA) patients, focusing on immunomodulatory effects. RA MSCs showed decreased proliferative activity and aberrant migration capacity. No significant differences were observed in cytokine profiles between RA and control MSCs. The effects of RA MSCs on proliferation of peripheral blood mononuclear cells (PBMCs) and distribution of specific CD4+ T cell subtypes (Th17, Treg, and Tfh cells) were investigated. RA MSCs appeared to be indistinguishable from controls in suppressing PBMC proliferation, decreasing the proportion of Tfh cells, and inducing the polarization of Treg cells. However, the capacity to inhibit Th17 cell polarization was impaired in RA MSCs, which was related to the low expression of CCL2 in RA MSCs after coculture with CD4+ T cells. These findings indicated that RA MSCs display defects in several important biological activities, especially the capacity to inhibit Th17 cell polarization. These functionally impaired MSCs may contribute to the development of RA disease.
doi:10.1155/2015/284215
PMCID: PMC4397051  PMID: 25918734
24.  Effect of Rhizosphere Enzymes on Phytoremediation in PAH-Contaminated Soil Using Five Plant Species 
PLoS ONE  2015;10(3):e0120369.
A pot experiment was performed to study the effectiveness of remediation using different plant species and the enzyme response involved in remediating PAH-contaminated soil. The study indicated that species Echinacea purpurea, Festuca arundinacea Schred, Fire Phoenix (a combined F. arundinacea), and Medicago sativa L. possess the potential for remediation in PAH-contaminated soils. The study also determined that enzymatic reactions of polyphenol oxidase (except Fire Phoenix), dehydrogenase (except Fire Phoenix), and urease (except Medicago sativa L.) were more prominent over cultivation periods of 60d and 120d than 150d. Urease activity of the tested species exhibited prominently linear negative correlations with alkali-hydrolyzable nitrogen content after the tested plants were cultivated for 150d (R2 = 0.9592). The experiment also indicated that alkaline phosphatase activity in four of the five tested species (Echinacea purpurea, Callistephus chinensis, Festuca arundinacea Schred and Fire Phoenix) was inhibited during the cultivation process (at 60d and 120d). At the same time, the study determined that the linear relationship between alkaline phosphatase activity and effective phosphorus content in plant rhizosphere soil exhibited a negative correlation after a growing period of 120d (R2 = 0.665). Phytoremediation of organic contaminants in the soil was closely related to specific characteristics of particular plant species, and the catalyzed reactions were the result of the action of multiple enzymes in the plant rhizosphere soil.
doi:10.1371/journal.pone.0120369
PMCID: PMC4378954  PMID: 25822167
25.  Ulinastatin Reduces the Resistance of Liver Cancer Cells to Epirubicin by Inhibiting Autophagy 
PLoS ONE  2015;10(3):e0120694.
During chemotherapy, drug resistance caused by autophagy remains a major challenge to successful treatment of cancer patients. The purpose of this study is to show that ulinastatin (UTI), a trypsin inhibitor, could reduce the resistance of liver cancer cells to chemotherapeutic agent epirubicin (EPI). We achieved this conclusion by analyzing the effect of EPI alone or UTI plus EPI on SMMC-7721 and MHCC-LM3 liver cancer cells. We also generated an EPI-resistant liver cancer cell line (MHCC-LM3er cells), and found that UTI could sensitize the LM3er cells to EPI. Autophagy usually functions to protect cancer cells during chemotherapy. Our study showed that UTI inhibited the autophagy induced by EPI in liver cancer cells, which promoted apoptosis, and therefore, reduced the resistance of the cancer cells to EPI. Further studies showed that the UTI-mediated inhibition on autophagy was achieved by inhibiting transcriptional factor nuclear factor-κB (NF-κB) signaling pathway. To verify our results in vivo, we injected MHCC-LM3 liver cancer cells or EPI-resistant LM3er cells into mice, and found that EPI could only effectively inhibit the growth of tumor in MHCC-LM3 cell-injected mice, but not in LM3er cell-injected mice. However, when UTI was also administered, the growth of tumor was inhibited in the MHCC-LM3er cell-injected mice as well. Our results suggest that UTI may be used in combination with anti-cancer drugs, such as EPI, to improve the outcome of cancer therapy.
doi:10.1371/journal.pone.0120694
PMCID: PMC4376693  PMID: 25815885

Results 1-25 (232)