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1.  Sodium tanshinone IIA silate as an add-on therapy in patients with unstable angina pectoris 
Journal of Thoracic Disease  2014;6(12):1794-1799.
To investigate whether sodium tanshinone IIA silate (STS) as an add-on therapy to conventional treatment may provide additional benefits for patients with unstable angina pectoris (UAP) and is associated with changes in profiles of serum inflammatory factors.
Eighty patients diagnosed with UAP were randomly divided into two groups for the 2-week treatment. The control group received conventional therapy, while the treatment group was given intravenous STS (0.06 mg in 250 mL, once daily) as an add-on therapy to the conventional medications. The therapeutic efficacy and changes in serum levels of several inflammatory cytokines, including monocyte chemotactic protein 1 (MCP-1), tumor necrosis factor alpha (TNF-α), peroxisome proliferator-activated receptor (PPAR-γ), and high-sensitivity C-reactive protein (hs-CRP) from baseline were determined and compared between the two group.
The clinical symptoms of all patients in both groups were improved after treatment. The overall rate of effectiveness was 97.5% in the treatment group vs. 80.0% in the control group. Serum levels of MCP-1, TNF-α, and hs-CRP levels were significantly reduced in both groups (P<0.01), whereas the reduction was greater in patients receiving additional STS (P<0.05). PPAR-γ was significantly elevated in both groups (P<0.01).
STS in combination with conventional treatment may be associated with better outcomes in patients with UAP.
PMCID: PMC4283346  PMID: 25589975
Unstable angina pectoris (UAP); sodium tanshinone IIA silate (STS); monocyte chemotactic protein 1 (MCP-1); tumor necrosis factor alpha (TNF-α); peroxisome proliferator-activated receptor (PPAR-γ); high-sensitivity C-reactive protein (hs-CRP)
2.  Effects of Treg cells and IDO on human epithelial ovarian cancer cells under hypoxic conditions 
Molecular Medicine Reports  2014;11(3):1708-1714.
The aim of the present study was to explore the effect of hypoxia on ovarian cancer. A total of 6 samples were analyzed: SKOV3-IP cells (ovarian cancer cell line); SKOV3-IP and regulatory T (Treg) cells; SKOV3-IP and cytotoxic T lymphocytes (CTLs); SKOV3-IP and natural killer (NK) cells; SKOV3-IP co-cultured with CTLs and Treg cells; and SKOV3-IP co-cultured with Treg cells and NK cells. The expression of indoleamine 2,3-dioxygenase (IDO) was detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. An enzyme-linked immunosorbent assay (ELISA) was used to detect the concentration of transforming growth factor-β (TGF-β), interferon-γ (IFN-γ), interleukin-2 (IL-2), interleukin-10 (IL-10), and perforin. Moreover, ovarian cancer cell apoptosis and invasive ability were examined using flow cytometry and a Transwell chamber assay. IDO expression was significantly reduced in hypoxia and enhanced by Treg cells. Treg cells inhibited the IL-2, IFN-γ and perforin secretion, and significantly (P<0.05) increased the IL-10 and TGF-β levels. The effects of Treg cells were enhanced with prolongation of the cell exposure to hypoxic conditions. In addition, Treg cells attenuated the promotive effect of CTLs and NK cells on cancer cell apoptosis. In addition, Treg cells significantly increased the SKOV3-IP invasive ability (P=0.00109) under hypoxic conditions. Our results suggest that IDO and Treg cells may serve as important therapeutic targets for patients with ovarian cancer.
PMCID: PMC4270340  PMID: 25376937
epithelial ovarian cancer; IDO; regulatory T cells; hypoxia
3.  Serum Metabonomic Analysis of Protective Effects of Curcuma aromatica Oil on Renal Fibrosis Rats 
PLoS ONE  2014;9(9):e108678.
Curcuma aromatica oil is a traditional herbal medicine demonstrating protective and anti-fibrosis activities in renal fibrosis patients. However, study of its mechanism of action is challenged by its multiple components and multiple targets that its active agent acts on.
Methodology/Principal Findings
Nuclear magnetic resonance (NMR)-based metabonomics combined with clinical chemistry and histopathology examination were performed to evaluate intervening effects of Curcuma aromatica oil on renal interstitial fibrosis rats induced by unilateral ureteral obstruction. The metabolite levels were compared based on integral values of serum 1H NMR spectra from rats on 3, 7, 14, and 28 days after the medicine administration. Time trajectory analysis demonstrated that metabolic profiles of the agent-treated rats were restored to control levels after 7 days of dosage. The results confirmed that the agent would be an effective anti-fibrosis medicine in a time-dependent manner, especially in early renal fibrosis stage. Targeted metabolite analysis showed that the medicine could lower levels of lipid, acetoacetate, glucose, phosphorylcholine/choline, trimethylamine oxide and raise levels of pyruvate, glycine in the serum of the rats. Serum clinical chemistry and kidney histopathology examination dovetailed well with the metabonomics data.
The results substantiated that Curcuma aromatica oil administration can ameliorate renal fibrosis symptoms by inhibiting some metabolic pathways, including lipids metabolism, glycolysis and methylamine metabolism, which are dominating targets of the agent working in vivo. This study further strengthens the novel analytical approach for evaluating the effect of traditional herbal medicine and elucidating its molecular mechanism.
PMCID: PMC4181651  PMID: 25265289
4.  The Role of N-Acetyltransferase 8 in Mesenchymal Stem Cell-Based Therapy for Liver Ischemia/Reperfusion Injury in Rats 
PLoS ONE  2014;9(7):e103355.
To evaluate the impact of mesenchymal stem cells (MSCs) against hepatic I/R injury and explore the role of N-acetyltransferase 8 (NAT8) in the process.
We investigated the potential of injected MSCs systemically via the tail vein in healing injuried liver of the SD rat model of 70% hepatic I/R injury by measuring the biochemical and pathologic alterations. Subsequently, we evaluated the expression levels of NAT8 by western blotting in vivo. Concurrently, hydrogen peroxide (H2O2)-induced apoptosis in the human normal liver cell line L02 was performed in vitro to evaluate the protective effects of MSC conditioned medium (MSC-CM) on L02 cells. In addition, we downregulated and upregulated NAT8 expression in L02 cells and induced apoptosis by using H2O2 to study the protective role of NAT8.
MSCs implantation led to a significant reduced liver enzyme levels, an advanced protection in the histopathological findings of the acutely injured liver and a significantly lower percentage of TUNEL-positive cells, which were increased after I/R injury. In vitro assays, MSC-CM inhibited hepatocyte apoptosis induced by H2O2. Moreover, overexpression or downregulation of NAT8 prevented or aggravated hepatocyte apoptosis induced by H2O2, respectively.
MSC transplantation provides support to the I/R-injured liver by inhibiting hepatocellular apoptosis and stimulating NAT8 regeneration.
PMCID: PMC4109999  PMID: 25057902
5.  Effects of Chinese Medicine Tong xinluo on Diabetic Nephropathy via Inhibiting TGF-β1-Induced Epithelial-to-Mesenchymal Transition 
Diabetic nephropathy (DN) is a major cause of chronic kidney failure and characterized by interstitial and glomeruli fibrosis. Epithelial-to-mesenchymal transition (EMT) plays an important role in the pathogenesis of DN. Tong xinluo (TXL), a Chinese herbal compound, has been used in China with established therapeutic efficacy in patients with DN. To investigate the molecular mechanism of TXL improving DN, KK-Ay mice were selected as models for the evaluation of pathogenesis and treatment in DN. In vitro, TGF-β1 was used to induce EMT. Western blot (WB), immunofluorescence staining, and real-time polymerase chain reaction (RT-PCR) were applied to detect the changes of EMT markers in vivo and in vitro, respectively. Results showed the expressions of TGF-β1 and its downstream proteins smad3/p-smad3 were greatly reduced in TXL group; meantime, TXL restored the expression of smad7. As a result, the expressions of collagen IV (Col IV) and fibronectin (FN) were significantly decreased in TXL group. In vivo, 24 h-UAER (24-hour urine albumin excretion ratio) and BUN (blood urea nitrogen) were decreased and Ccr (creatinine clearance ratio) was increased in TXL group compared with DN group. In summary, the present study demonstrates that TXL successfully inhibits TGF-β1-induced epithelial-to-mesenchymal transition in DN, which may account for the therapeutic efficacy in TXL-mediated renoprotection.
PMCID: PMC4016864  PMID: 24864150
6.  Proteome-wide quantification and characterization of oxidation-sensitive cysteines in pathogenic bacteria 
Cell host & microbe  2013;13(3):358-370.
Thiol group oxidation of active and allosteric cysteines is a widespread regulatory post-translational protein modification. Pathogenic bacteria, including Pseudomonas aeruginosa and Staphylococcus aureus, use regulatory cysteine oxidation to respond to and overcome reactive oxygen species (ROS) encountered in the host environment. To obtain a proteome-wide view of oxidation-sensitive cysteines in these two pathogens, we employed a competitive activity-based protein profiling approach to globally quantify hydrogen peroxide (H2O2) reactivity with cysteines across bacterial proteomes. We identified ~200 proteins containing H2O2-sensitive cysteines, including metabolic enzymes, transcription factors, and uncharacterized proteins. Further biochemical and genetic studies identified an oxidation-responsive cysteine in the master quorum sensing regulator LasR, and redox-regulated activities for acetaldehyde dehydrogenase ExaC, arginine deiminase ArcA, and glyceraldehyde 3-phosphate dehydrogenase GAPDH. Taken together, our data indicate that pathogenic bacteria exhibit a complex, multi-layered response to ROS that includes the rapid adaption of metabolic pathways to oxidative stress challenge.
PMCID: PMC3652280  PMID: 23498960
7.  Development of a direct competitive ELISA for the detection of Mycoplasma bovis infection based on a monoclonal antibody of P48 protein 
Mycoplasma bovis (M. bovis) is a major, but often overlooked, pathogen documented to cause respiratory disease, mastitis, and arthritis in cattle throughout China since 2008. Here, we report the development of a direct competitive enzyme-linked immunosorbent assay (Dc-ELISA) to detect M. bovis antibody.
We used a recombinant P48 protein and monoclonal antibody (mAb) 10E. MAb 10E, prepared against the recombinant P48 protein of M. bovis, identified all M. bovis strains with no cross-reactivity with other related pathogens. Coating micro plates with P48 protein instead of whole M. bovis cells as well as the use of mAb 10E produced a specific and sensitive Dc-ELISA for M. bovis antibody detection with a cut-off percent inhibition (PI) value of 32%. Compared with two commercial indirect ELISA (i-ELISA) kits, our Dc-ELISA offered a higher positive detection rate in 165 clinical bovine serum samples.
A rapid, sensitive, and reliable serological diagnosis method was developed for M. bovis, which can facilitate M. bovis surveillance, assisting researchers in understanding the ecology and epidemiology of M. bovis.
PMCID: PMC3942108  PMID: 24533468
Direct competitive ELISA; Mycoplasma bovis; Monoclonal antibody
8.  Studies Based on Preparation, Physical Characteristics, and Cellular Pharmacological Activities of Thin PLGA Film Loaded with Geniposide 
In this primary study, thin polylactic-co-glycolic acid (PLGA) film loaded with geniposide was first prepared and demonstrated on both physical and pharmacological aspects for its potential application on drug-eluting vascular stents. Physical parameters of geniposide-loaded thin film, such as crystal structure, molecular spectral characteristics, and release behavior in the whole process were detected. From X-Ray diffraction, the characteristic peak of crystal geniposide disappeared on geniposide-loaded PLGA film (GLPF) after it formed, which meant there was no agglomeration phenomenon, as geniposide was distributed in the form of single molecule. According to scanning electron microscopy (SEM) figure, the GLPF was more flat and uniform with better compactness. It inferred that release behavior of geniposide at the early stage (0~15 d) was in the form of free diffusion. Carrier PLGA began to degrade 15 days later, so the residual geniposide was also dissolved. Cellular pharmacological effects of geniposide on endothelial cells (ECs) and smooth muscle cells (SMCs) were also demonstrated on GLPF. 5% and 10% (w/w) geniposide-loaded PLGA (60 : 40) membrane indicated its significant effect on ECs promotion and SMCs inhibition. All provided feasible evidences for the development of new geniposide-coating vascular stent using PLGA as carrier.
PMCID: PMC3944942  PMID: 24693321
9.  Identification of Novel Immunogenic Proteins from Mycoplasma bovis and Establishment of an Indirect ELISA Based on Recombinant E1 Beta Subunit of the Pyruvate Dehydrogenase Complex 
PLoS ONE  2014;9(2):e88328.
The pathogen Mycoplasma bovis (M. bovis) is a major cause of respiratory disease, mastitis, and arthritis in cattle. Screening the key immunogenic proteins and updating rapid diagnostic techniques are necessary to the prevention and control of M. bovis infection. In this study, 19 highly immunogenic proteins from M. bovis strain PD were identified using 2-dimensional gel electrophoresis, immunoblotting and MALDI-TOF/TOF MS. Of these 19 proteins, pyruvate dehydrogenase E1 component beta subunit (PDHB) showed excellent immune reactivity and repeatability. PDHB was found to be conserved in different M. bovis isolates, as indicated by Western blot analysis. On the basis of these results, a rPDHB-based indirect ELISA (iELISA) was established for the detection of serum antibodies using prokaryotically expressed recombinant PDHB protein as the coating antigen. The specificity analysis result showed that rPDHB-based iELISA did not react with other pathogens assessed in our study except M. agalactiae (which infects sheep and goats). Moreover, 358 serum samples from several disease-affected cattle feedlots were tested using this iELISA system and a commercial kit, which gave positive rates of 50.8% and 39.9%, respectively. The estimated Kappa agreement coefficient between the two methods was 0.783. Notably, 39 positive serum samples that had been missed by the commercial kit were all found to be positive by Western blot analysis. The detection rate of rPDHB-based iELISA was significantly higher than that of the commercial kit at a serum dilution ratio of 1∶5120 to 1∶10,240 (P<0.05). Taken together, these results provide important information regarding the novel immunogenic proteins of M. bovis. The established rPDHB-based iELISA may be suitable for use as a new method of antibody detection in M. bovis.
PMCID: PMC3919759  PMID: 24520369
10.  Modification of Pectin and Hemicellulose Polysaccharides in Relation to Aril Breakdown of Harvested Longan Fruit 
To investigate the modification of cell wall polysaccharides in relation to aril breakdown in harvested longan fruit, three pectin fractions (WSP, water soluble pectin; CSP, CDTA-soluble pectin; ASP, alkali soluble pectin) and one hemicellulose fraction (4 M KOH-SHC, 4 M KOH-soluble hemicellulose) were extracted, and their contents, monosaccharide compositions and molecular weights were evaluated. As aril breakdown intensified, CSP content increased while ASP and 4 M KOH-SHC contents decreased, suggesting the solubilization and conversion of cell wall components. Furthermore, the molar percentage of arabinose (Ara), as the main component of the side-chains, decreased largely in CSP and ASP while that of rhamnose (Rha), as branch point for the attachment of neutral sugar side chains, increased during aril breakdown. Analysis of (Ara + Gal)/Rha ratio showed that the depolymerization of CSP and ASP happened predominantly in side-chains formed of Ara residues. For 4 M KOH-SHC, more backbones were depolymerized during aril breakdown. Moreover, it was found that the molecular weights of CSP, ASP and 4 M KOH-SHC polysaccharides tended to decrease as aril breakdown intensified. These results suggest that both enhanced depolymerization and structural modifications of polysaccharides in the CSP, ASP and 4 M KOH-SHC fractions might be responsible for aril breakdown of harvested longan fruit.
PMCID: PMC3876050  PMID: 24287911
aril breakdown; cell wall degradation; pectin; matrix glycan; depolymerization
11.  Robust and specific ratiometric biosensing using a copper-free clicked quantum dot–DNA aptamer sensor† †Electronic supplementary information (ESI) available: Details on the synthesis, purification and characterisation of the DHLA–PEG600–N3, cyclooctyne–DNA, and QD–TBA20 conjugates as well as all supporting figures and tables. See DOI: 10.1039/c3nr02897fClick here for additional data file.  
Nanoscale  2013;5(21):10307-10315.
A robust and copper-free clicked quantum dot–DNA sensor for sensitive and ratiometric detection of specific DNA and protein targets at the pM level is reported.
We report herein the successful preparation of a compact and functional CdSe–ZnS core–shell quantum dot (QD)–DNA conjugate via highly efficient copper-free “click chemistry” (CFCC) between a dihydro-lipoic acid–polyethylene glycol–azide (DHLA–PEG–N3) capped QD and a cyclooctyne modified DNA. This represents an excellent balance between the requirements of high sensitivity, robustness and specificity for the QD-FRET (Förster resonance energy transfer) based sensor as confirmed by a detailed FRET analysis on the QD–DNA conjugate, yielding a relatively short donor–acceptor distance of ∼5.8 nm. We show that this CFCC clicked QD–DNA conjugate is not only able to retain the native fluorescence quantum yield (QY) of the parent DHLA–PEG–N3 capped QD, but also well-suited for robust and specific biosensing; it can directly quantitate, at the pM level, both labelled and unlabelled complementary DNA probes with a good SNP (single-nucleotide polymorphism) discrimination ability in complex media, e.g. 10% human serum via target-binding induced FRET changes between the QD donor and the dye acceptor. Furthermore, this sensor has also been successfully exploited for the detection, at the pM level, of a specific protein target (thrombin) via the encoded anti-thrombin aptamer sequence in the QD–DNA conjugate.
PMCID: PMC3814187  PMID: 24056667
12.  Adaptive Modulation of Adult Brain Gray and White Matter to High Altitude: Structural MRI Studies 
PLoS ONE  2013;8(7):e68621.
The aim of this study was to investigate brain structural alterations in adult immigrants who adapted to high altitude (HA). Voxel-based morphometry analysis of gray matter (GM) volumes, surface-based analysis of cortical thickness, and Tract-Based Spatial Statistics analysis of white matter fractional anisotropy (FA) based on MRI images were conducted on 16 adults (20–22 years) who immigrated to the Qinghai-Tibet Plateau (2300–4400 m) for 2 years. They had no chronic mountain sickness. Control group consisted of 16 matched sea level subjects. A battery of neuropsychological tests was also conducted. HA immigrants showed significantly decreased GM volumes in the right postcentral gyrus and right superior frontal gyrus, and increased GM volumes in the right middle frontal gyrus, right parahippocampal gyrus, right inferior and middle temporal gyri, bilateral inferior ventral pons, and right cerebellum crus1. While there was some divergence in the left hemisphere, surface-based patterns of GM changes in the right hemisphere resembled those seen for VBM analysis. FA changes were observed in multiple WM tracts. HA immigrants showed significant impairment in pulmonary function, increase in reaction time, and deficit in mental rotation. Parahippocampal and middle frontal GM volumes correlated with vital capacity. Superior frontal GM volume correlated with mental rotation and postcentral GM correlated with reaction time. Paracentral lobule and frontal FA correlated with mental rotation reaction time. There might be structural modifications occurred in the adult immigrants during adaptation to HA. The changes in GM may be related to impaired respiratory function and psychological deficits.
PMCID: PMC3712920  PMID: 23874692
13.  Structural Modulation of Brain Development by Oxygen: Evidence on Adolescents Migrating from High Altitude to Sea Level Environment 
PLoS ONE  2013;8(7):e67803.
The present study aimed to investigate structural modulation of brain by high level of oxygen during its peak period of development. Voxel-based morphometry analysis of gray matter (GM) and white matter (WM) volumes and Tract-Based Spatial Statistics analysis of WM fractional anisotropy (FA) and mean diffusion (MD) based on MRI images were carried out on 21 Tibetan adolencents (15–18 years), who were born and raised in Qinghai-Tibetan Plateau (2900–4700 m) and have lived at sea level (SL) in the last 4 years. The control group consisted of matched Tibetan adolescents born and raised at high altitude all the time. SL immigrants had increased GM volume in the left insula, left inferior parietal gyrus, and right superior parietal gyrus and decreased GM in the left precentral cortex and multiple sites in cerebellar cortex (left lobule 8, bilateral lobule 6 and crus 1/2). Decreased WM volume was found in the right superior frontal gyrus in SL immigrants. SL immigrants had higher FA and lower MD at multiple sites of WM tracts. Moreover, we detected changes in ventilation and circulation. GM volume in cerebellum lobule 8 positively correlated with diastolic pressure, while GM volume in insula positively correlated vital capacity and hypoxic ventilatory response. Our finding indicate that the structural modulations of GM by high level of oxygen during its peak period of development are related to respiratory and circulatory regulations, while the modulation in WM mainly exhibits an enhancement in myelin maturation.
PMCID: PMC3706444  PMID: 23874449
14.  Impacts of Nitrate and Nitrite on Physiology of Shewanella oneidensis 
PLoS ONE  2013;8(4):e62629.
Shewanella oneidensis exhibits a remarkable versatility in anaerobic respiration, which largely relies on its diverse respiratory pathways. Some of these are expressed in response to the existence of their corresponding electron acceptors (EAs) under aerobic conditions. However, little is known about respiration and the impact of non-oxygen EAs on the physiology of the microorganism when oxygen is present. Here we undertook a study to elucidate the basis for nitrate and nitrite inhibition of growth under aerobic conditions. We discovered that nitrate in the form of NaNO3 exerts its inhibitory effects as a precursor to nitrite at low concentrations and as an osmotic-stress provider (Na+) at high concentrations. In contrast, nitrite is extremely toxic, with 25 mM abolishing growth completely. We subsequently found that oxygen represses utilization of all EAs but nitrate. To order to utilize EAs with less positive redox potential, such as nitrite and fumarate, S. oneidensis must enter the stationary phase, when oxygen respiration becomes unfavorable. In addition, we demonstrated that during aerobic respiration the cytochrome bd oxidase confers S. oneidensis resistance to nitrite, which likely functions via nitric oxide (NO).
PMCID: PMC3633839  PMID: 23626841
15.  Vaccinia virus leads to ATG12–ATG3 conjugation and deficiency in autophagosome formation 
Autophagy  2011;7(12):1434-1447.
The interactions between viruses and cellular autophagy have been widely reported. On the one hand, autophagy is an important innate immune response against viral infection. On the other hand, some viruses exploit the autophagy pathway for their survival and proliferation in host cells. Vaccinia virus is a member of the family of Poxviridae which includes the smallpox virus. The biogenesis of vaccinia envelopes, including the core envelope of the immature virus (IV), is not fully understood. In this study we investigated the possible interaction between vaccinia virus and the autophagy membrane biogenesis machinery. Massive LC3 lipidation was observed in mouse fibroblast cells upon vaccinia virus infection. Surprisingly, the vaccinia virus induced LC3 lipidation was shown to be independent of ATG5 and ATG7, as the atg5 and atg7 null mouse embryonic fibroblasts (MEFs) exhibited the same high levels of LC3 lipidation as compared with the wild-type MEFs. Mass spectrometry and immunoblotting analyses revealed that the viral infection led to the direct conjugation of ATG3, which is the E2-like enzyme required for LC3-phosphoethanonamine conjugation, to ATG12, which is a component of the E3-like ATG12–ATG5-ATG16 complex for LC3 lipidation. Consistently, ATG3 was shown to be required for the vaccinia virus induced LC3 lipidation. Strikingly, despite the high levels of LC3 lipidation, subsequent electron microscopy showed that vaccinia virus-infected cells were devoid of autophagosomes, either in normal growth medium or upon serum and amino acid deprivation. In addition, no autophagy flux was observed in virus-infected cells. We further demonstrated that neither ATG3 nor LC3 lipidation is crucial for viral membrane biogenesis or viral proliferation and infection. Together, these results indicated that vaccinia virus does not exploit the cellular autophagic membrane biogenesis machinery for their viral membrane production. Moreover, this study demonstrated that vaccinia virus instead actively disrupts the cellular autophagy through a novel molecular mechanism that is associated with aberrant LC3 lipidation and a direct conjugation between ATG12 and ATG3.
PMCID: PMC3327614  PMID: 22024753
ATG12; ATG3; autophagy; LC3 lipidation; vaccinia virus
16.  ER stress–mediated autophagy promotes Myc-dependent transformation and tumor growth 
The Journal of Clinical Investigation  2012;122(12):4621-4634.
The proto-oncogene c-Myc paradoxically activates both proliferation and apoptosis. In the pathogenic state, c-Myc–induced apoptosis is bypassed via a critical, yet poorly understood escape mechanism that promotes cellular transformation and tumorigenesis. The accumulation of unfolded proteins in the ER initiates a cellular stress program termed the unfolded protein response (UPR) to support cell survival. Analysis of spontaneous mouse and human lymphomas demonstrated significantly higher levels of UPR activation compared with normal tissues. Using multiple genetic models, we demonstrated that c-Myc and N-Myc activated the PERK/eIF2α/ATF4 arm of the UPR, leading to increased cell survival via the induction of cytoprotective autophagy. Inhibition of PERK significantly reduced Myc-induced autophagy, colony formation, and tumor formation. Moreover, pharmacologic or genetic inhibition of autophagy resulted in increased Myc-dependent apoptosis. Mechanistically, we demonstrated an important link between Myc-dependent increases in protein synthesis and UPR activation. Specifically, by employing a mouse minute (L24+/–) mutant, which resulted in wild-type levels of protein synthesis and attenuation of Myc-induced lymphomagenesis, we showed that Myc-induced UPR activation was reversed. Our findings establish a role for UPR as an enhancer of c-Myc–induced transformation and suggest that UPR inhibition may be particularly effective against malignancies characterized by c-Myc overexpression.
PMCID: PMC3533536  PMID: 23143306
17.  Brain cell apoptosis and enhancement of nervous excitability in pregnant rats with high plasma levels of homocysteine☆ 
Neural Regeneration Research  2012;7(28):2199-2205.
Hyperhomocysteinemia is an important risk factor for preeclampsia-eclampsia. This study established a pregnant rat model of hyperhomocysteinemia, in which blood plasma homocysteine concentrations were twice or three times greater than that of normal pregnant rats. TUNEL revealed an increase in the number of apoptotic cells in the frontal cortex of pregnant rats with hyperhomocysteinemia. In addition, immunohistochemical staining detected activated nuclear factor-κB-positve cells in the frontal cortex. Reverse transcription-PCR detected that mRNA expression of the anti-apoptotic gene bcl-2 diminished in the frontal cortex. In situ hybridization and western blotting revealed that N-methyl-D-aspartate receptor 1 mRNA and protein expression was upregulated in the frontal cortex and hippocampus. These results indicate that hyperhomocysteinemia can induce brain cell apoptosis, increase nerve excitability, and promote the occurrence of preeclampsia in pregnant rats.
PMCID: PMC4268719  PMID: 25538740
hyperhomocysteinemia; homocysteine; preeclampsia; frontal cortex; N-methyl-D-aspartate receptor; nerve excitability; cell apoptosis; pregnancy; brain; neural regeneration
18.  Inhibition of Glycogen Synthase Kinase 3β Ameliorates D-GalN/LPS-Induced Liver Injury by Reducing Endoplasmic Reticulum Stress-Triggered Apoptosis 
PLoS ONE  2012;7(9):e45202.
Glycogen synthase kinase 3β(GSK3β) is a ubiquitous serine-threonine protein kinase that participates in numerous cellular processes and disease pathophysiology. We aimed to determine therapeutic potential of GSK3β inhibition and its mechanism in a well-characterized model of lipopolysaccharide (LPS)-induced model of acute liver failure (ALF).
In a murine ALF model induced by D-GalN(700 mg/kg)/LPS(10 µg/kg), we analyzed GSK3β mechanisms using a specific chemical inhibitor, SB216763, and detected the role of endoplasmic reticulum stress (ERS). Mice were administered SB216763 at 2 h before or after D-GalN/LPS injection, respectively, and then sacrificed 6 h after D-GalN/LPS treatment to evaluate its prophylactic and therapeutic function. The lethality rate, liver damage, ERS, cytokine expression, MAP kinase, hepatocyte apoptosis and expression of TLR 4 were evaluated, respectively. Whether the inhibition of GSK3β activation protected hepatocyte from ERS-induced apoptosis was investigated in vitro.
Principal Findings
GSK3β became quickly activated (dephosphorylated) upon D-GalN/LPS exposure. Administration of SB216763 not only ameliorated liver injury, as evidenced by reduced transaminase levels, and well-preserved liver architecture, but also decreased lethality. Moreover, GSK3β inhibition resulted in down-regulation of pro-apoptotic proteins C/EBP–homologous protein(CHOP) and caspase-12, which are related to ERS. To further demonstrate the role of ERS, we found that GSK3β inhibition protected hepatocyte from ERS-induced cell death. GSK3β inhibition down-regulated the MAPK pathways, reduced expression of inflammatory cytokines and decreased expression of TLR4.
Our findings demonstrate the key function of GSK3β signaling in the pathophysiology of ALF, especially in regulating the ERS, and provide a rationale for targeting GSK3β as a potential therapeutic strategy to ameliorate ALF.
PMCID: PMC3461002  PMID: 23028846
19.  A cohort study evaluating paraaortic lymphadenectomy in endometrial cancer 
Oncology Letters  2012;4(6):1361-1365.
The current study sought to assess the role of paraaortic lymphadenectomy (LNE) in females with endometrial cancer. A retrospective analysis of patients diagnosed with endometrial cancer of stage IA to II preoperatively, between 2009 and 2011 was conducted. Patients were included who had suffered from endometrial cancer without preoperative adjuvant therapy and who underwent hysterectomy plus systematic pelvic LNE and paraaortic LNE by laparoscopy or laparotomy. A total of 54 patients who underwent surgery for preoperative endometrial cancer were selected. All patients underwent LNE. The incidences of pelvic and paraaortic lymph node metastases were 11.1% (6/54) and 7.4% (4/54), with a total positive lymph node rate of 14.8% (8/54). In addition, among the 8 positive cases, 5 patients underwent laparotomy and 3 underwent laparoscopy; 3 cases were classified as stage I and 5 as stage II preoperatively. Of these, 7 patients were identified with pathology-related risk factors, including low differentiation or clear cell adenocarcinoma postoperatively. Discordance of pathological differentiation between the pre- and postoperative stages reached 57.1% (4/7). The results revealed the high occurrence of positive lymph nodes in endometrial cancer which demonstrate the importance of systematic LNE. Additonally, no severe complications were caused by LNE besides lymph cysts. In summary, it is neccesary to perform LNE, particularly the removal of the paraaortic lymph node, in patients with endometrial cancers in order to improve postoperative therapy. Laparoscopy has similar surgical effects as laparotomy, but has a number of advantages.
PMCID: PMC3506758  PMID: 23205136
lymphadenectomy; endometrial cancer; paraaortic
20.  Identification and characterization of small non-coding RNAs from Chinese fir by high throughput sequencing 
BMC Plant Biology  2012;12:146.
Small non-coding RNAs (sRNAs) play key roles in plant development, growth and responses to biotic and abiotic stresses. At least four classes of sRNAs have been well characterized in plants, including repeat-associated siRNAs (rasiRNAs), microRNAs (miRNAs), trans-acting siRNAs (tasiRNAs) and natural antisense transcript-derived siRNAs. Chinese fir (Cunninghamia lanceolata) is one of the most important coniferous evergreen tree species in China. No sRNA from Chinese fir has been described to date.
To obtain sRNAs in Chinese fir, we sequenced a sRNA library generated from seeds, seedlings, leaves, stems and calli, using Illumina high throughput sequencing technology. A comprehensive set of sRNAs were acquired, including conserved and novel miRNAs, rasiRNAs and tasiRNAs. With BLASTN and MIREAP we identified a total of 115 conserved miRNAs comprising 40 miRNA families and one novel miRNA with precursor sequence. The expressions of 16 conserved and one novel miRNAs and one tasiRNA were detected by RT-PCR. Utilizing real time RT-PCR, we revealed that four conserved and one novel miRNAs displayed developmental stage-specific expression patterns in Chinese fir. In addition, 209 unigenes were predicted to be targets of 30 Chinese fir miRNA families, of which five target genes were experimentally verified by 5' RACE, including a squamosa promoter-binding protein gene, a pentatricopeptide (PPR) repeat-containing protein gene, a BolA-like family protein gene, AGO1 and a gene of unknown function. We also demonstrated that the DCL3-dependent rasiRNA biogenesis pathway, which had been considered absent in conifers, existed in Chinese fir. Furthermore, the miR390-TAS3-ARF regulatory pathway was elucidated.
We unveiled a complex population of sRNAs in Chinese fir through high throughput sequencing. This provides an insight into the composition and function of sRNAs in Chinese fir and sheds new light on land plant sRNA evolution.
PMCID: PMC3462689  PMID: 22894611
Chinese fir; miRNA; rasiRNA; tasiRNA; Cunninghamia lanceolata
21.  Grey and white matter abnormalities in chronic obstructive pulmonary disease: a case–control study 
BMJ Open  2012;2(2):e000844.
The irreversible airflow limitation characterised by chronic obstructive pulmonary disease (COPD) causes a decrease in the oxygen supply to the brain. The aim of the present study was to investigate brain structural damage in COPD.
Retrospective case–control study. Patients with COPD and healthy volunteers were recruited. The two groups were matched in age, gender and educational background.
A hospital and a number of communities: they are all located in southern Fujian province, China.
25 stable patients and 25 controls were enrolled from December 2009 to May 2011.
Using voxel-based morphometry and tract-based spatial statistics based on MRI to analyse grey matter (GM) density and white matter fractional anisotropy (FA), respectively, and a battery of neuropsychological tests were performed.
Patients with COPD (vs controls) showed decreased GM density in the limbic and paralimbic structures, including right gyrus rectus, left precentral gyrus, bilateral anterior and middle cingulate gyri, bilateral superior temporal gyri, bilateral anterior insula extending to Rolandic operculum, bilateral thalamus/pulvinars and left caudate nucleus. Patients with COPD (vs controls) had decreased FA values in the bilateral superior corona radiata, bilateral superior and inferior longitudinal fasciculus, bilateral optic radiation, bilateral lingual gyri, left parahippocampal gyrus and fornix. Lower FA values in these regions were associated with increased radial diffusivity and no changes of longitudinal diffusivity. Patients with COPD had poor performances in the Mini-Mental State Examination, figure memory and visual reproduction. GM density in some decreased regions in COPD had positive correlations with arterial blood Po2, negative correlations with disease duration and also positive correlations with visual tasks.
The authors demonstrated that COPD exhibited loss of regional GM accompanied by impairment of white matter microstructural integrity, which was associated with disease severity and may underlie the pathophysiological and psychological changes of COPD.
Article summary
Article focus
Decreased oxygen supply to brain may cause neuronal damage in COPD. However, the damage remains largely uninvestigated.
Key messages
We found that COPD extends to the brain, with the loss of regional cortical grey matter accompanied by impairment in the white matter microstructural integrity.
Our findings would be help for clinical therapy of COPD.
Strengths and limitations of this study
Multiple analyses were used based on MR images. The statistic power for FA analysis was weak.
PMCID: PMC3341600  PMID: 22535793
22.  Transcriptome-wide identification and characterization of miRNAs from Pinus densata 
BMC Genomics  2012;13:132.
MicroRNAs (miRNAs) play key roles in diverse developmental processes, nutrient homeostasis and responses to biotic and abiotic stresses. The biogenesis and regulatory functions of miRNAs have been intensively studied in model angiosperms, such as Arabidopsis thaliana, Oryza sativa and Populus trichocarpa. However, global identification of Pinus densata miRNAs has not been reported in previous research.
Here, we report the identification of 34 conserved miRNAs belonging to 25 miRNA families from a P. densata mRNA transcriptome database using local BLAST and MIREAP programs. The primary and/or precursor sequences of 29 miRNAs were further confirmed by RT-PCR amplification and subsequent sequencing. The average value of the minimal folding free energy indexes of the 34 miRNA precursors was 0.92. Nineteen (58%) mature miRNAs began with a 5' terminal uridine residue. Analysis of miRNA precursors showed that 19 mature miRNAs were novel members of 14 conserved miRNA families, of which 17 miRNAs were further validated by subcloning and sequencing. Using real-time quantitative RT-PCR, we found that the expression levels of 7 miRNAs were more than 2-fold higher in needles than in stems. In addition, 72 P. densata mRNAs were predicted to be targets of 25 miRNA families. Four target genes, including a nodal modulator 1-like protein gene, two GRAS family transcription factor protein genes and one histone deacetylase gene, were experimentally verified to be the targets of 3 P. densata miRNAs, pde-miR162a, pde-miR171a and pde-miR482a, respectively.
This study led to the discovery of 34 conserved miRNAs comprising 25 miRNA families from Pinus densata. These results lay a solid foundation for further studying the regulative roles of miRNAs in the development, growth and responses to environmental stresses in P. densata.
PMCID: PMC3347991  PMID: 22480283
Pinus densata; miRNA; Transcriptome
23.  Co-Inoculation with Rhizobia and AMF Inhibited Soybean Red Crown Rot: From Field Study to Plant Defense-Related Gene Expression Analysis 
PLoS ONE  2012;7(3):e33977.
Soybean red crown rot is a major soil-borne disease all over the world, which severely affects soybean production. Efficient and sustainable methods are strongly desired to control the soil-borne diseases.
Principal Findings
We firstly investigated the disease incidence and index of soybean red crown rot under different phosphorus (P) additions in field and found that the natural inoculation of rhizobia and arbuscular mycorrhizal fungi (AMF) could affect soybean red crown rot, particularly without P addition. Further studies in sand culture experiments showed that inoculation with rhizobia or AMF significantly decreased severity and incidence of soybean red crown rot, especially for co-inoculation with rhizobia and AMF at low P. The root colony forming unit (CFU) decreased over 50% when inoculated by rhizobia and/or AMF at low P. However, P addition only enhanced CFU when inoculated with AMF. Furthermore, root exudates of soybean inoculated with rhizobia and/or AMF significantly inhibited pathogen growth and reproduction. Quantitative RT-PCR results indicated that the transcripts of the most tested pathogen defense-related (PR) genes in roots were significantly increased by rhizobium and/or AMF inoculation. Among them, PR2, PR3, PR4 and PR10 reached the highest level with co-inoculation of rhizobium and AMF.
Our results indicated that inoculation with rhizobia and AMF could directly inhibit pathogen growth and reproduction, and activate the plant overall defense system through increasing PR gene expressions. Combined with optimal P fertilization, inoculation with rhizobia and AMF could be considered as an efficient method to control soybean red crown rot in acid soils.
PMCID: PMC3307780  PMID: 22442737
24.  Transcriptional Profiling of ESTs from the Biocontrol Fungus Chaetomium cupreum 
The Scientific World Journal  2012;2012:340565.
Comparative analysis was applied to two cDNA/ESTs libraries (C1 and C2) from Chaetomium cupreum. A total of 5538 ESTs were sequenced and assembled into 2162 unigenes including 585 contigs and 1577 singletons. BlastX analysis enabled the identification of 1211 unigenes with similarities to sequences in the public databases. MFS monosaccharide transporter was found as the gene expressed at the highest level in library C2, but no expression in C1. The majority of unigenes were library specific. Comparative analysis of the ESTs further revealed the difference of C. cupreum in gene expression and metabolic pathways between libraries. Two different sequences similar to the 48-KDa endochitinase and 46-KDa endochitinase were identified in libraries C1 and C2, respectively.
PMCID: PMC3289965  PMID: 22448129
25.  Automatic categorization of diverse experimental information in the bioscience literature 
BMC Bioinformatics  2012;13:16.
Curation of information from bioscience literature into biological knowledge databases is a crucial way of capturing experimental information in a computable form. During the biocuration process, a critical first step is to identify from all published literature the papers that contain results for a specific data type the curator is interested in annotating. This step normally requires curators to manually examine many papers to ascertain which few contain information of interest and thus, is usually time consuming. We developed an automatic method for identifying papers containing these curation data types among a large pool of published scientific papers based on the machine learning method Support Vector Machine (SVM). This classification system is completely automatic and can be readily applied to diverse experimental data types. It has been in use in production for automatic categorization of 10 different experimental datatypes in the biocuration process at WormBase for the past two years and it is in the process of being adopted in the biocuration process at FlyBase and the Saccharomyces Genome Database (SGD). We anticipate that this method can be readily adopted by various databases in the biocuration community and thereby greatly reducing time spent on an otherwise laborious and demanding task. We also developed a simple, readily automated procedure to utilize training papers of similar data types from different bodies of literature such as C. elegans and D. melanogaster to identify papers with any of these data types for a single database. This approach has great significance because for some data types, especially those of low occurrence, a single corpus often does not have enough training papers to achieve satisfactory performance.
We successfully tested the method on ten data types from WormBase, fifteen data types from FlyBase and three data types from Mouse Genomics Informatics (MGI). It is being used in the curation work flow at WormBase for automatic association of newly published papers with ten data types including RNAi, antibody, phenotype, gene regulation, mutant allele sequence, gene expression, gene product interaction, overexpression phenotype, gene interaction, and gene structure correction.
Our methods are applicable to a variety of data types with training set containing several hundreds to a few thousand documents. It is completely automatic and, thus can be readily incorporated to different workflow at different literature-based databases. We believe that the work presented here can contribute greatly to the tremendous task of automating the important yet labor-intensive biocuration effort.
PMCID: PMC3305665  PMID: 22280404

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