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1.  Reactivation of the homeotic tumor suppressor gene CDX2 by 5-aza-2′-deoxycytidine-induced demethylation inhibits cell proliferation and induces caspase-independent apoptosis in gastric cancer cells 
The DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-aza-CdR) is widely used as an anticancer drug for the treatment of leukemia and solid tumors. Gastric cancer (GC) patients who were positive for caudal type homeobox transcription factor 2 (CDX2) expression showed a higher survival rate compared with those who were CDX2 negative, which suggests that CDX2 performs a tumor suppressor role. However, the molecular mechanisms leading to the inactivation of CDX2 remain unclear. In the present study we demonstrated that the expression levels of CDX2 and DNA methyltransferase enzyme 1 (DNMT1) mRNA were significantly higher in GC compared with distal non-cancerous tissue. The expression of CDX2 mRNA was significantly correlated with Lauren classification, TNM stage and lymph node metastasis. DNMT1 mRNA expression was significantly correlated with TNM stage, pathological differentiation and lymph node metastasis. The expression of CDX2 mRNA was inversely correlated with that of DNMT1 mRNA in GC. Hypermethylation of the CDX2 gene promoter region, extremely low expression levels of CDX2 mRNA and no expression of CDX2 protein were the characteristics observed in MKN-45 and SGC-7901 GC cell lines. Following the treatment of MKN-45 cells with 5-aza-CdR, the hypermethylated CDX2 gene promoter region was demethylated and expression of CDX2 was upregulated, while DNMT1 expression was downregulated. Furthermore, a concentration- and time-dependent growth inhibition as well as increased apoptosis were observed. Caspase-3, −8 and −9 activities increased in a concentration-dependent manner following exposure to different concentrations of 5-aza-CdR. Therefore, our data show that the overexpression of DNMT1 and methylation of the CDX2 gene promoter region is likely to be responsible for CDX2 silencing in GC. 5-Aza-CdR may effectively induce re-expression of the CDX2 gene, inhibit cell proliferation and enhance the caspase-independent apoptosis of MKN-45 cells in vitro.
PMCID: PMC3570199  PMID: 23408490
gastric neoplasms; caudal type homeobox transcription factor 2; DNA methylation; DNA methyltransferase enzyme 1; proliferation; apoptosis
2.  Relationship between the downregulation of HLA class I antigen and clinicopathological significance in gastric cancer 
AIM: To discuss the expression of human leukocyte antigen (HLA) class I antigens in gastric cancer and correlate these with pathologic type and TNM stage.
METHODS: The expression of HLA class I antigen was detected by immunohistochemistry in 185 specimens of gastric cancer, 20 gastric cancer specimens with lymphatic metastasis and 22 controls of normal gastric mucosa using four monoclonal antibodies.
RESULTS: The expression of HLA class I antigen (B/C locus) was significantly downregulated in gastric cancer and in lymphatic metastasis than that in normal gastric mucosa (χ2 = 7.712, P<0.05). The expression of other HLA class I antigens was also downregulated, but the change was slight. There was no relationship between the downregulation of HLA class I antigen and that of β2m and LMP2. The expression of HLA class I (B/C locus) was statistically correlated with pathologic stage in gastric adenocarcinoma (χ2 = 4.164, P<0.05).
CONCLUSION: The expression of HLA class I antigen (B/C locus) was obviously downregulated in gastric cancer and in lymphatic metastasis. This abnormal expression would provide the tumor cells with a way to avoid immunological recognition.
PMCID: PMC4315976  PMID: 15962390
Downregulation; Gastric cancer
3.  Crystal Structures of PI3Kα Complexed with PI103 and Its Derivatives: New Directions for Inhibitors Design 
ACS Medicinal Chemistry Letters  2013;5(2):138-142.
The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays important roles in cell proliferation, growth, and survival. Hyperactivated PI3K is frequently found in a wide variety of human cancers, validating it as a promising target for cancer therapy. We determined the crystal structure of the human PI3Kα–PI103 complex to unravel molecular interactions. Based on the structure, substitution at the R1 position of the phenol portion of PI103 was demonstrated to improve binding affinity via forming a new H-bond with Lys802 at the bottom of the ATP catalytic site. Interestingly, the crystal structure of the PI3Kα–9d complex revealed that the flexibility of Lys802 can also induce additional space at the catalytic site for further modification. Thus, these crystal structures provide a molecular basis for the strong and specific interactions and demonstrate the important role of Lys802 in the design of novel PI3Kα inhibitors.
PMCID: PMC4027628  PMID: 24900786
PI3K; PI103; crystal structure; drug design; cancer therapy
4.  Stress Response and Tolerance of Zea mays to CeO2 Nanoparticles: Cross Talk among H2O2, Heat Shock Protein and Lipid Peroxidation 
ACS nano  2012;6(11):9615-9622.
The rapid development of nanotechnology will inevitably release nanoparticles (NPs) into the environment with unidentified consequences. In addition, the potential toxicity of CeO2 NPs to plants, and the possible transfer into the food chain, are still unknown. Corn plants (Zea mays) were germinated and grown in soil treated with CeO2 NPs at 400 or 800 mg/kg. Stress related parameters, such as: H2O2, catalase (CAT) and ascorbate peroxidase (APX) activity, heat shock protein 70 (HSP 70), lipid peroxidation, cell death and leaf gas exchange were analyzed at 10, 15, and 20 days post germination. Confocal laser scanning microscopy was used to image H2O2 distribution in corn leaves. Results showed that the CeO2 NP treatments increased accumulation of H2O2, up to day 15, in phloem, xylem, bundle sheath cells, and epidermal cells of shoots. The CAT and APX activities were also increased in the corn shoot, concomitant with the H2O2 levels. Both 400 and 800 mg/kg CeO2 NPs triggered the up regulation of the HSP 70 in roots, indicating a systemic stress response. None of the CeO2 NPs increased the level of thiobarbituric acid reacting substances, indicating that no lipid peroxidation occurred. CeO2 NPs, at both concentrations, did not induce ion leakage in either roots or shoots, suggesting membrane integrity was not compromised. Leaf net photosynthetic rate, transpiration, and stomatal conductance were not affected by CeO2 NPs. Our results suggest that the CAT, APX and HSP 70 might help the plants defend against CeO2 NPs induced oxidative injury and survive NP exposure.
PMCID: PMC4326050  PMID: 23050848
CeO2 NPs; Zea mays; H2O2; stress enzymes; cell death; photosynthesis
5.  TBX6 Null Variants and a Common Hypomorphic Allele in Congenital Scoliosis 
The New England journal of medicine  2015;372(4):341-350.
Congenital scoliosis is a common type of vertebral malformation. Genetic susceptibility has been implicated in congenital scoliosis.
We evaluated 161 Han Chinese persons with sporadic congenital scoliosis, 166 Han Chinese controls, and 2 pedigrees, family members of which had a 16p11.2 deletion, using comparative genomic hybridization, quantitative polymerase-chain-reaction analysis, and DNA sequencing. We carried out tests of replication using an additional series of 76 Han Chinese persons with congenital scoliosis and a multi-center series of 42 persons with 16p11.2 deletions.
We identified a total of 17 heterozygous TBX6 null mutations in the 161 persons with sporadic congenital scoliosis (11%); we did not observe any null mutations in TBX6 in 166 controls (P<3.8×10−6). These null alleles include copy-number variants (12 instances of a 16p11.2 deletion affecting TBX6) and single-nucleotide variants (1 nonsense and 4 frame-shift mutations). However, the discordant intrafamilial phenotypes of 16p11.2 deletion carriers suggest that heterozygous TBX6 null mutation is insufficient to cause congenital scoliosis. We went on to identify a common TBX6 haplotype as the second risk allele in all 17 carriers of TBX6 null mutations (P<1.1×10−6). Replication studies involving additional persons with congenital scoliosis who carried a deletion affecting TBX6 confirmed this compound inheritance model. In vitro functional assays suggested that the risk haplotype is a hypomorphic allele. Hemivertebrae are characteristic of TBX6-associated congenital scoliosis.
Compound inheritance of a rare null mutation and a hypomorphic allele of TBX6 accounted for up to 11% of congenital scoliosis cases in the series that we analyzed.
PMCID: PMC4326244  PMID: 25564734
6.  Better operative outcomes achieved with the prone jackknife vs. lithotomy position during abdominoperineal resection in patients with low rectal cancer 
Lithotomy (LT) and prone jackknife positions (PJ) are routinely used for abdominoperineal resection (APR). The present study compared the clinical, pathological, and oncological outcomes of PJ-APR vs. LT-APR in low rectal cancer patients in order to confirm which position will provide more benefits to patients undergoing APR.
This is a retrospective study of consecutive patients with low rectal cancer who underwent curative APR between January 2002 and December 2011. Patients were matched 1:2 (PJ-APR = 74 and LT-APR = 37 patients) based on gender and age. Perioperative data, postoperative outcomes, and survival were compared between the two approaches.
Hospital stay was shorter with PJ-APR compared with LT-APR (P < 0.05). Compared with LT-APR, duration of anesthesia (234 ± 50.8 vs. 291 ± 69 min, P = 0.022) and surgery (183 ± 44.8 vs. 234 ± 60 min, P = 0.016) was shorter with PJ-APR, and estimated blood losses were smaller (549 ± 218 vs. 674 ± 350 mL, P < 0.001). Blood transfusions were required in 37.8% of LT-APR patients and in 8.1% of PJ-APR patients (P < 0.001). There was no difference in the distribution of N stages (P = 0.27). Median follow-up was 47.1 (13.6–129.7) months. Postoperative complications were reported by fewer patients after PJ-APR compared with LT-APR (14.9% vs. 32.4%, P = 0.030). There were no significant differences in overall survival, disease-free survival, local recurrence, and distant metastasis (P > 0.05).
The PJ position provided a better exposure for low rectal cancer and had a lower operative risk and complication rates than LT-APR. However, there was no difference in rectal cancer prognosis between the two approaches. PJ-APR might be a better choice for patients with low rectal cancer.
PMCID: PMC4331390
Rectal cancer; Abdominoperineal resection; Lithotomy position; Prone jackknife position
7.  Association between levonorgestrel emergency contraception and the risk of ectopic pregnancy: a multicenter case-control study 
Scientific Reports  2015;5:8487.
Cases of ectopic pregnancy (EP) following levonorgestrel emergency contraception (LNG-EC) failure have been reported continuously, but whether there is an association between EP risk and LNG-EC is unclear. We concluded a case-control study to explore this association by recruiting 2,411 EP patients as case group, and 2,416 women with intrauterine pregnancy and 2,419 non-pregnant women as control groups. Odds ratios (ORs) and their 95% confidential intervals (CIs) were calculated and adjusted for potential confounding factors. Previous use of LNG-EC was not correlated with the EP. Compared to women who did not use contraceptives, current use of LNG-EC reduced the risk for intrauterine pregnancy (Adjusted OR [AOR] = 0.20, 95%CI: 0.14–0.27), but did not increase the risk for EP (AOR2 = 1.04, 95%CI: 0.76–1.42). Furthermore, compared to women who did not have further act of intercourse, women with unprotected further act of intercourse were at a higher risk of EP (AOR1 = 2.35, 95%CI: 1.17–4.71), and women with repeated use of LNG-EC for further intercourse during the same cycle was also associated with a higher risk for EP (AOR1 = 3.08, 95%CI: 1.09–8.71; AOR2 = 2.49, 95%CI: 1.00–6.19). A better understanding of the risk of EP following LNG-EC failure can optimize LNG-EC use and thus reduce the risk of EP.
PMCID: PMC4325579  PMID: 25674909
8.  Fingerprint analysis of processed Rhizoma Chuanxiong by high-performance liquid chromatography coupled with diode array detection 
Chinese Medicine  2015;10:2.
Rhizoma Chuanxiong (RC) is the dried rhizome of Ligusticum chuanxiong Hort., and various types of processed Rhizoma Chuanxiong (PRC) are widely used in China. However, quality assurance and quality control of these processed medicines remain challenging. This study aims to investigate the chemical compositions of various PRC preparations by a high-performance liquid chromatography (HPLC) coupled with diode array detection (DAD) method.
A HPLC-DAD method with validation was developed for PRC samples. Seven batches of plant samples from two processing methods, stir-frying and steaming, were analyzed by the HPLC-DAD method. Common peaks in PRC chromatograms were chosen to calculate their relative retention time (RRT) and relative peak area (RPA), and similarity analyses of the chromatographic fingerprints were conducted by Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine software (Version 2004 A).
In the 24-h stability test, the relative standard deviation for the RRT and RPA was less than 0.07% and 2.57%, respectively. The precision was less than 0.08% for the RRT and 2.48% for the RPA. The repeatability for the RRT and RPA was less than 0.03% and 2.64%, respectively. The similarities between the seven PRC batches were range from 0.956 to 0.990. After stir-frying or steaming, the amount of ferulic acid in PRC was much higher than that in the raw material.
The fingerprint analysis of PRC by different processing methods was feasible by HPLC-DAD.
PMCID: PMC4336472
Rhizoma Chuanxiong; HPLC-DAD; Ligusticum chuanxiong; Fingerprint; Processing
9.  EIF3i Promotes Colon Oncogenesis by Regulating COX-2 Protein Synthesis and β-Catenin Activation 
Oncogene  2013;33(32):4156-4163.
Translational control of gene expression has recently been recognized as an important mechanism controlling cell proliferation and oncogenesis and it mainly occurs in the initiation step of protein synthesis that involves multiple eukaryotic initiation factors (eIFs). Many eIFs have been found to have aberrant expression in human tumors and the aberrant expression may contribute to oncogenesis. However, how these previously considered house-keeping proteins are potentially oncogenic remains elusive. In this study, we investigated the expression of eIF3i in human colon cancers, tested its contribution to colon oncogenesis, and determined the mechanism of eIF3i action in colon oncogenesis. We found that eIF3i expression was up-regulated in both human colon adenocarcinoma and adenoma polyps as well as in model inducible colon tumorigenic cell lines. Over-expression of ectopic eIF3i in intestinal epithelial cells causes oncogenesis by directly up-regulating synthesis of COX-2 protein and activates the β-catenin/TCF4 signaling pathway that mediates the oncogenic function of eIF3i. Together, we conclude that eIF3i is a proto-oncogene that drives colon oncogenesis by translationally up-regulating COX-2 and activating β-catenin signaling pathway. These findings imply that protooncogenic eIFs likely exert their tumorigenic function by regulating/altering the synthesis level of down-stream tumor suppressor or oncogenes.
PMCID: PMC3962800  PMID: 24056964
eIF3i; COX-2; β-catenin; translational control; colon cancer; RNA-binding
10.  Does the Arcto-Tertiary Biogeographic Hypothesis Explain the Disjunct Distribution of Northern Hemisphere Herbaceous Plants? The Case of Meehania (Lamiaceae) 
PLoS ONE  2015;10(2):e0117171.
Despite considerable progress, many details regarding the evolution of the Arcto-Tertiary flora, including the timing, direction, and relative importance of migration routes in the evolution of woody and herbaceous taxa of the Northern Hemisphere, remain poorly understood. Meehania (Lamiaceae) comprises seven species and five subspecies of annual or perennial herbs, and is one of the few Lamiaceae genera known to have an exclusively disjunct distribution between eastern Asia and eastern North America. We analyzed the phylogeny and biogeographical history of Meehania to explore how the Arcto-Tertiary biogeographic hypothesis and two possible migration routes explain the disjunct distribution of Northern Hemisphere herbaceous plants. Parsimony and Bayesian inference were used for phylogenetic analyses based on five plastid sequences (rbcL, rps16, rpl32-trnH, psbA-trnH, and trnL-F) and two nuclear (ITS and ETS) gene regions. Divergence times and biogeographic inferences were performed using Bayesian methods as implemented in BEAST and S-DIVA, respectively. Analyses including 11 of the 12 known Meehania taxa revealed incongruence between the chloroplast and nuclear trees, particularly in the positions of Glechoma and Meehania cordata, possibly indicating allopolyploidy with chloroplast capture in the late Miocene. Based on nrDNA, Meehania is monophyletic, and the North American species M. cordata is sister to a clade containing the eastern Asian species. The divergence time between the North American M. cordata and the eastern Asian species occurred about 9.81 Mya according to the Bayesian relaxed clock methods applied to the combined nuclear data. Biogeographic analyses suggest a primary role of the Arcto-Tertiary flora in the study taxa distribution, with a northeast Asian origin of Meehania. Our results suggest an Arcto-Tertiary origin of Meehania, with its present distribution most probably being a result of vicariance and southward migrations of populations during climatic oscillations in the middle Miocene with subsequent migration into eastern North America via the Bering land bridge in the late Miocene.
PMCID: PMC4319762  PMID: 25658699
11.  The role of Sec3p in secretory vesicle targeting and exocyst complex assembly 
Molecular Biology of the Cell  2014;25(23):3813-3822.
The exocyst has been speculated to mediate the tethering of secretory vesicles to the plasma membrane. However, there has been no direct experimental evidence for this notion. An ectopic targeting strategy is used to provide experimental support for this model and investigate the regulators of exocyst assembly and vesicle targeting.
During membrane trafficking, vesicular carriers are transported and tethered to their cognate acceptor compartments before soluble N-ethylmaleimide–sensitive factor attachment protein (SNARE)-mediated membrane fusion. The exocyst complex was believed to target and tether post-Golgi secretory vesicles to the plasma membrane during exocytosis. However, no definitive experimental evidence is available to support this notion. We developed an ectopic targeting assay in yeast in which each of the eight exocyst subunits was expressed on the surface of mitochondria. We find that most of the exocyst subunits were able to recruit the other members of the complex there, and mistargeting of the exocyst led to secretion defects in cells. On the other hand, only the ectopically located Sec3p subunit is capable of recruiting secretory vesicles to mitochondria. Our assay also suggests that both cytosolic diffusion and cytoskeleton-based transport mediate the recruitment of exocyst subunits and secretory vesicles during exocytosis. In addition, the Rab GTPase Sec4p and its guanine nucleotide exchange factor Sec2p regulate the assembly of the exocyst complex. Our study helps to establish the role of the exocyst subunits in tethering and allows the investigation of the mechanisms that regulate vesicle tethering during exocytosis.
PMCID: PMC4230786  PMID: 25232005
12.  NLRP1 inflammasome is activated in patients with medial temporal lobe epilepsy and contributes to neuronal pyroptosis in amygdala kindling-induced rat model 
Temporal lobe epilepsy (TLE) is often characterized pathologically by severe neuronal loss in the hippocampus. Understanding the mechanisms of neuron death is key to preventing the neurodegeneration associated with TLE. However, the involvement of neuronal loss to the epileptogenic process has yet to be fully determined. Recent studies have shown that the activation of NLRP1 can generate a functional caspase-1-containing inflammasome in vivo to drive the proinflammatory programmed cell death termed ‘pyroptosis’, which has a key role in the pathogenesis of neurological disorders. To the best of our knowledge, there are no reported studies that performed detailed identification and validation of NLRP1 inflammasome during the epileptogenic process.
We first compared expression of NLRP1 and caspase-1 in resected hippocampus from patients with intractable mesial temporal lobe epilepsy (mTLE) with that of matched control samples. To further examine whether the activation of NLRP1 inflammasome contributes to neuronal pyroptosis, we employed a nonviral strategy to knock down the expression of NLRP1 and caspase-1 in the amygdala kindling-induced rat model. Proinflammatory cytokines levels and hippocampal neuronal loss were evaluated after 6 weeks of treatment in these NLRP1 or caspase-1 deficiency TLE rats.
Western blotting detected upregulated NLRP1 levels and active caspase-1 in mTLE patients in comparison to those levels seen in the controls, suggesting a role for this inflammasome in mTLE. Moreover, we employed direct in vivo infusion of nonviral small interfering RNA to knockdown NLRP1 or caspase-1 in the amygdala kindling-induced rat model, and discovered that these NLRP1 or caspase-1 silencing rats resulted in significantly reduced neuronal pyroptosis.
Our data suggest that NLRP1/caspase-1 signaling participates in the seizure-induced degenerative process in humans and in the animal model of TLE and points to the silencing of NLRP1 inflammasome as a promising strategy for TLE therapy.
Electronic supplementary material
The online version of this article (doi:10.1186/s12974-014-0233-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4314732  PMID: 25626361
NLRP1; pyroptosis; inflammasome; Caspase-1; temporal lobe epilepsy
13.  Effect of hydrogen sulfide on inflammatory cytokines in acute myocardial ischemia injury in rats 
Hydrogen sulfide (H2S) is believed to be involved in numerous physiological and pathophysiological processes, and now it is recognized as the third endogenous signaling gasotransmitter, following nitric oxide and carbon monoxide; however, the effects of H2S on inflammatory factors in acute myocardial ischemia injury in rats have not been clarified. In the present study, sodium hydrosulfide (NaHS) was used as the H2S donor. Thirty-six male Sprague Dawley rats were randomly divided into five groups: Sham, ischemia, ischemia + low-dose (0.78 mg/kg) NaHS, ischemia + medium-dose (1.56 mg/kg) NaHS, ischemia + high-dose (3.12 mg/kg) NaHS and ischemia + propargylglycine (PPG) (30 mg/kg). The rats in each group were sacrificed 6 h after the surgery for sample collection. Compared with the ischemia group, the cardiac damage in the rats in the ischemia + NaHS groups was significantly reduced, particularly in the high-dose group; in the ischemia + PPG group, the myocardial injury was aggravated compared with that in the ischemia group. Compared with the ischemia group, the levels of interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) in the serum of rats in the ischemia + medium- and high-dose NaHS groups were significantly reduced, and the expression of intercellular adhesion molecule-1 (ICAM-1) mRNA and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) protein in the myocardial tissues of rats was significantly reduced. In the ischemia + PPG group, the TNF-α, IL-1β and IL-6 levels in the serum were significantly increased, the expression of ICAM-1 mRNA was increased, although without a significant difference, and the expression of NF-κB was increased. The findings of the present study provide novel evidence for the dual effects of H2S on acute myocardial ischemia injury via the modulation of inflammatory factors.
PMCID: PMC4316979  PMID: 25667680
hydrogen sulfide; acute myocardial ischemia; rat; inflammatory factor
14.  Centralized isolation of Helicobacter pylori from multiple centers and transport condition influences 
AIM: To evaluate the efficacy of centralized culture and possible influencing factors.
METHODS: From January 2010 to July 2012, 66452 patients with suspected Helicobacter pylori (H. pylori) infection from 26 hospitals in Zhejiang and Jiangsu Provinces in China underwent gastrointestinal endoscopy. Gastric mucosal biopsies were taken from the antrum for culture. These biopsies were transported under natural environmental temperature to the central laboratory in Hangzhou city and divided into three groups based on their transport time: 5, 24 and 48 h. The culture results were reported after 72 h and the positive culture rates were analyzed by a χ2 test. An additional 5736 biopsies from H. pylori-positive patients (5646 rapid urease test-positive and 90 14C-urease breath test-positive) were also cultured for quality control in the central laboratory setting.
RESULTS: The positive culture rate was 31.66% (21036/66452) for the patient samples and 71.72% (4114/5736) for the H. pylori-positive quality control specimens. In the 5 h transport group, the positive culture rate was 30.99% (3865/12471), and 32.84% (14960/45553) in the 24 h transport group. In contrast, the positive culture rate declined significantly in the 48 h transport group (26.25%; P < 0.001). During transportation, the average natural temperature increased from 4.67 to 29.14 °C, while the positive culture rate declined from 36.67% (1462/3987) to 24.12% (1799/7459). When the temperature exceeded 24 °C, the positive culture rate decreased significantly, especially in the 48 h transport group (23.17%).
CONCLUSION: Transportation of specimens within 24 h and below 24 °C is reasonable and acceptable for centralized culture of multicenter H. pylori samples.
PMCID: PMC4299348  PMID: 25624729
Centralized isolation; Helicobacter pylori; Influencing factor; Multiple centers; Personalized treatment
15.  Inhibition of oxidative phosphorylation for enhancing citric acid production by Aspergillus niger 
The spore germination rate and growth characteristics were compared between the citric acid high-yield strain Aspergillus niger CGMCC 5751 and A. niger ATCC 1015 in media containing antimycin A or DNP. We inferred that differences in citric acid yield might be due to differences in energy metabolism between these strains. To explore the impact of energy metabolism on citric acid production, the changes in intracellular ATP, NADH and NADH/NAD+ were measured at various fermentation stages. In addition, the effects of antimycin A or DNP on energy metabolism and citric acid production was investigated by CGMCC 5751.
By comparing the spore germination rate and the extent of growth on PDA plates containing antimycin A or DNP, CGMCC 5751 was shown to be more sensitive to antimycin A than ATCC 1015. The substrate-level phosphorylation of CGMCC 5751 was greater than that of ATCC 1015 on PDA plates with DNP. DNP at tested concentrations had no apparent effect on the growth of CGMCC 5751. There were no apparent effects on the mycelial morphology, the growth of mycelial pellets or the dry cell mass when 0.2 mg L-1 antimycin A or 0.1 mg L-1 DNP was added to medium at the 24-h time point. The concentrations of intracellular ATP, NADH and NADH/NAD+ of CGMCC 5751 were notably lower than those of ATCC 1015 at several fermentation stages. Moreover, at 96 h of fermentation, the citric acid production of CGMCC 5751 reached up to 151.67 g L-1 and 135.78 g L-1 by adding 0.2 mg L-1 antimycin A or 0.1 mg L-1 DNP, respectively, at the 24-h time point of fermentation. Thus, the citric acid production of CGMCC 5751 was increased by 19.89% and 7.32%, respectively.
The concentrations of intracellular ATP, NADH and NADH/NAD+ of the citric acid high-yield strain CGMCC 5751 were notably lower than those of ATCC 1015. The excessive ATP has a strong inhibitory effect on citric acid accumulation by A. niger. Increasing NADH oxidation and appropriately reducing the concentration of intracellular ATP can accelerate glycolysis and the TCA cycle to enhance citric acid yield.
Electronic supplementary material
The online version of this article (doi:10.1186/s12934-015-0190-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4320542  PMID: 25592678
Citric acid; Aspergillus niger; Oxidative phosphorylation inhibitor; Uncoupler of oxidative phosphorylation; Energy metabolism
16.  Autoantibodies in Chinese patients with chronic hepatitis B: Prevalence and clinical associations 
AIM: To investigate the prevalence of autoantibodies and their associations with clinical features in Chinese patients with chronic hepatitis B (CHB).
METHODS: A total of 325 Chinese patients with CHB were enrolled in this retrospective, hospital-based study. Patients with chronic hepatitis C (CHC), autoimmune hepatitis (AIH), or primary biliary cirrhosis (PBC) were included, with healthy donors acting as controls. A panel of autoantibodies that serologically define AIH and PBC was tested by indirect immunofluorescence assay and line immunoassay. The AIH-related autoantibody profile included homogeneous anti-nuclear antibodies (ANA-H), smooth-muscle antibodies, anti-liver kidney microsome type 1, anti-liver cytosolic antigen type 1, and anti-soluble liver antigen/liver pancreas; the PBC-related antibodies were characterized by ANA-nuclear dots/membranous rim-like, anti-mitochondrial antibodies-M2 (AMA-M2), anti-BPO (recombinant antigen targeted by AMA-M2), anti-Sp100, anti-promyelocytic leukemia protein (anti-PML), and anti-gp210. The dichotomization of clustering was used to unequivocally designate the AIH or PBC profiles for each case. Anti-Ro52 antibodies were also tested.
RESULTS: The prevalence of any autoantibody in CHB amounted to 58.2%, which was similar to the 66.2% prevalence in CHC, significantly higher than the 6.7% in the healthy controls (P < 0.001), and lower than the 100% found in AIH and PBC (P = 0.004 and P < 0.001, respectively). There were more anti-PML and anti-gp210 antibodies among the CHB patients than the CHC patients (11.1% vs 0%, P = 0.003; 12.6% vs 0%, P < 0.001, respectively). The prevalence and titer of AMA, anti-BPO, anti-PML, and anti-gp210 were higher in PBC than in those with CHB. Among the CHB patients, the prevalence of ANA, especially ANA-H, was significantly lower in patients with compensated and decompensated cirrhosis compared with patients without cirrhosis. Thirty-eight cases of hepatocellular carcinoma (HCC) in CHB showed a significant difference compared with non-HCC patients in the prevalence of anti-PML (0% vs 12.5%, P = 0.013). Dichotomization of the autoantibodies revealed that the PBC profile was more prevalent in patients with CHB than in those with CHC, and that it was strongly correlated with both compensated and decompensated cirrhosis. In contrast, the prevalence of the AIH profile was significantly higher in non-cirrhosis patients with CHB than in those with compensated cirrhosis (18.5% vs 8.2%, P = 0.039). Moreover, the AIH profile was also closely associated with hepatitis B e-antigen positivity.
CONCLUSION: ANA-H could be an indicator of early-stage CHB. Dichotomizing the autoantibody profiles revealed that the PBC profile is strongly associated with cirrhosis in CHB.
PMCID: PMC4284347  PMID: 25574103
Autoantibodies; Chronic hepatitis B; Autoimmune hepatitis; Primary biliary cirrhosis; Cirrhosis; Hepatocellular carcinoma
17.  MiR-125a-3p Regulates Glioma Apoptosis and Invasion by Regulating Nrg1 
PLoS ONE  2015;10(1):e0116759.
The current study was designed to examine the functional role and mechanism of miR-125a-3p in glioma development. Quantitative RT-PCR was used to evaluate miR-125a-3p expression in 60 glioma cases of different malignant grades. Then, the clinic pathologic significance of miR-125a-3p expression was determined in combination with the prognosis of the patients. In addition, the effects and mechanisms of miR-125a-3p on the proliferation, apoptosis and invasion of glioma cells were further investigated. The results showed that the expression of miR-125a-3p was decreased significantly in most malignant glioma samples relative to normal brain tissues and glioma tissues of low-malignant degree. Further kaplan-meier survival analysis showed that the lower expression of miR-125a-3p was associated with a poor prognosis of GBM patients. Functional analysis showed that the reintroduction of miR-125a-3p into glioblastoma cell lines induces markedly the apoptosis and suppresses the proliferation and migration of glioblastoma cells in vitro and in vivo. Luciferase assay and Western blot analysis revealed that Nrg1 is a direct target of miR-125a-3p. Furthermore, an increased expression of Nrg1 could reverse the effects of overexpression of miR-125a-3p on the proliferation, apoptosis and migration of glioblastoma cells. These findings suggest that miR-125a-3p performed an important role in glioma development mediated by directly regulating the expression of Nrg1. This study also provides a potential target for diagnosis and treatment of malignant glioma.
PMCID: PMC4283963  PMID: 25560389
18.  Intracellular Poly(I:C) Initiated Gastric Adenocarcinoma Cell Apoptosis and Subsequently Ameliorated NK Cell Functions 
Natural killer (NK) cells are granular lymphocytic cells that exert essential functions in viral infection defense and tumor immune surveillance. However, the functions of NK cells were impaired in cancer patients. Polycytidylic acid [poly(I:C)] has been used as an immune adjuvant to improve innate and adaptive immune responses. In this study, intracellular poly(I:C) could trigger gastric adenocarcinoma cells apoptosis quickly. Meanwhile, the sensitivity of poly(I:C)-treated gastric adenocarcinoma cells to NK cell cytolysis was increased, concomitant with the elevated expression of MICA/B and Fas. Furthermore, the cytolytic activity of NK cells against tumor cells was augmented significantly by the supernatant from poly(I:C)-transfected tumor cells compared with NK cells treated by the supernatant from untreated tumor cells, as well as the proliferation and migration abilities of NK cells. In this process, the activating receptors and cytolysis-associated molecules of NK cells were up-regulated. Further investigation showed that type I interferon (IFN) produced by poly(I:C)-transfected gastric adenocarcinoma cells played an important role in this process. Our findings demonstrated that intracellular poly(I:C) not only triggered gastric adenocarcinoma cell apoptosis, but also enhanced NK responses via inducing type I IFN production by gastric adenocarcinoma cells. These functions make poly(I:C) a promising therapeutic medicine for gastric adenocarcinoma.
PMCID: PMC3887439  PMID: 24032591
19.  Enhanced PI3K p110α signaling confers acquired lapatinib resistance which can be effectively reversed by a p110α-selective PI3K inhibitor 
Molecular cancer therapeutics  2013;13(1):60-70.
While the HER2-targeting agents trastuzumab and lapatinib have improved the survival of patients with HER2-positive breast cancer, resistance to these targeted therapies is a major challenge. To investigate mechanisms of acquired lapatinib resistance, we generated acquired lapatinib resistance cell models by extended exposure of two HER2-positive breast cancer cell lines to lapatinib. Genomic and proteomic analyses revealed that lapatinib-resistant breast cancer cells gained additional PI3K activation through activating mutation in PI3K p110α and/or increasing protein expression of existing mutant p110α. p110α protein up-regulation in lapatinib-resistant cells occurred through gene amplification or post-transcriptional upregulation. Knockdown of p110α, but not p110β, the other PI3K catalytic subunit present in epithelial cells, inhibited proliferation of lapatinib-resistant cells, especially when combined with lapatinib. Lapatinib-resistant xenograft growth was inhibited persistently by combination treatment with the p110α-selective PI3K inhibitor BYL719 and lapatinib; the drug combination was also well-tolerated in mice. Mechanistically, the combination of lapatinib plus BYL719 more effectively inhibited Akt phosphorylation and, surprisingly, Erk phosphorylation, than either drug alone in the resistance model. These findings indicate that lapatinib resistance can occur through p110α protein upregulation-mediated, and/or mutation-induced, PI3K activation. Moreover, a combinatorial targeted therapy, lapatinib plus BYL719, effectively overcame lapatinib resistance in vivo and could be further tested in clinical trials. Finally, our findings indicate that p110β may be dispensable for lapatinib resistance in some cases. This allows the usage of p110α-specific PI3K inhibitors and thus may spare patients the toxicities of pan-PI3K inhibition to allow maximal dosage and efficacy.
PMCID: PMC3902650  PMID: 24249715
20.  Coenzyme Q10 Protects Astrocytes from ROS-Induced Damage through Inhibition of Mitochondria-Mediated Cell Death Pathway 
Coenzyme Q10 (CoQ10) acts by scavenging reactive oxygen species to protect neuronal cells against oxidative stress in neurodegenerative diseases. The present study was designed to examine whether CoQ10 was capable of protecting astrocytes from reactive oxygen species (ROS) mediated damage. For this purpose, ultraviolet B (UVB) irradiation was used as a tool to induce ROS stress to cultured astrocytes. The cells were treated with 10 and 25 μg/ml of CoQ10 for 3 or 24 h prior to the cells being exposed to UVB irradiation and maintained for 24 h post UVB exposure. Cell viability was assessed by MTT conversion assay. Mitochondrial respiration was assessed by respirometer. While superoxide production and mitochondrial membrane potential were measured using fluorescent probes, levels of cytochrome C (cyto-c), cleaved caspase-9, and caspase-8 were detected using Western blotting and/or immunocytochemistry. The results showed that UVB irradiation decreased cell viability and this damaging effect was associated with superoxide accumulation, mitochondrial membrane potential hyperpolarization, mitochondrial respiration suppression, cyto-c release, and the activation of both caspase-9 and -8. Treatment with CoQ10 at two different concentrations started 24 h before UVB exposure significantly increased the cell viability. The protective effect of CoQ10 was associated with reduction in superoxide, normalization of mitochondrial membrane potential, improvement of mitochondrial respiration, inhibition of cyto-c release, suppression of caspase-9. Furthermore, CoQ10 enhanced mitochondrial biogenesis. It is concluded that CoQ10 may protect astrocytes through suppression of oxidative stress, prevention of mitochondrial dysfunction, blockade of mitochondria-mediated cell death pathway, and enhancement of mitochondrial biogenesis.
PMCID: PMC4278255  PMID: 25552930
astrocyte; caspase; coenzyme Q10; mitochondrion; reactive oxygen species; ultraviolet.
21.  Fibroblast Growth Factors Stimulate Hair Growth through β-Catenin and Shh Expression in C57BL/6 Mice 
BioMed Research International  2015;2015:730139.
Growth factors are involved in the regulation of hair morphogenesis and cycle hair growth. The present study sought to investigate the hair growth promoting activities of three approved growth factor drugs, fibroblast growth factor 10 (FGF-10), acidic fibroblast growth factor (FGF-1), and basic fibroblast growth factor (FGF-2), and the mechanism of action. We observed that FGFs promoted hair growth by inducing the anagen phase in telogenic C57BL/6 mice. Specifically, the histomorphometric analysis data indicates that topical application of FGFs induced an earlier anagen phase and prolonged the mature anagen phase, in contrast to the control group. Moreover, the immunohistochemical analysis reveals earlier induction of β-catenin and Sonic hedgehog (Shh) in hair follicles of the FGFs-treated group. These results suggest that FGFs promote hair growth by inducing the anagen phase in resting hair follicles and might be a potential hair growth-promoting agent.
PMCID: PMC4313060
22.  Impact of chronic obstructive pulmonary diseases on left ventricular diastolic function in hospitalized elderly patients 
To evaluate the impact of chronic obstructive pulmonary disease (COPD) on left ventricular (LV) diastolic function in hospitalized elderly patients.
This was a case–control observational study of 148 consecutive hospitalized elderly patients (≥65 years old): 73 subjects without COPD as controls and 75 patients with COPD. Mild-to-moderate COPD was defined as stages 1 and 2, while severe and very severe COPD was defined as stages 3 and 4, according to the Global Initiative for Chronic Obstructive Lung Disease guidelines. Clinical characteristics and echocardiographic parameters were analyzed and compared.
Compared with the control group, patients with COPD had a higher frequency of LV diastolic dysfunction and heart failure with preserved ejection fraction. Smoking frequency, frequency of cerebrovascular diseases and diabetes, and serum N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels were higher in the COPD group (all P<0.05). COPD patients showed more abnormalities in diastolic function (E/e′: 11.51±2.50 vs 10.42±3.25, P=0.047), but no differences in systolic function and right ventricular function (all P>0.05). Patients with severe/very severe COPD showed no differences in LV diastolic function compared to patients with mild/moderate COPD (P>0.05), but serum NT-proBNP levels were higher in severe/very severe COPD (P<0.05).
Results suggest that early-stage COPD may have an impact on the LV diastolic function. Severe COPD mainly affected right ventricular function. In hospitalized elderly patients with COPD, LV diastolic dysfunction should be taken into account together with right ventricular function.
PMCID: PMC4279668  PMID: 25565790
chronic obstructive pulmonary disease; heart failure; left ventricular diastolic dysfunction; elderly; echocardiography
23.  Intracerebroventricular Transplantation of Ex Vivo Expanded Endothelial Colony-Forming Cells Restores Blood–Brain Barrier Integrity and Promotes Angiogenesis of Mice with Traumatic Brain Injury 
Journal of Neurotrauma  2013;30(24):2080-2088.
Endothelial progenitor cells (EPCs) play a key role in tissue repair and regeneration. Previous studies have shown a positive correlation between the number of circulating EPCs and clinical outcomes of patients with traumatic brain injury (TBI). A recent study has further shown that intravenous infusion of human umbilical cord blood-derived endothelial colony-forming cells (ECFCs) improves outcomes of mice subjected to experimental TBI. This follow-up study was designed to determine whether intracerebroventricular (i.c.v.) infusion of ECFCs, which may reduce systemic effects of these cells, could repair the blood–brain barrier (BBB) and promote angiogenesis of mice with TBI. Adult nude mice were exposed to fluid percussion injury and transplanted i.c.v. with ECFCs on day 1 post-TBI. These ECFCs were detected at the TBI zone 3 days after transplantation by SP-DiIC18(3) and fluorescence in situ hybridization. Mice with ECFCs transplant had reduced Evans blue extravasation and brain water content, increased expression of ZO-1 and claudin-5, and showed a higher expression of angiopoietin 1. Consistent with the previous report, mice with ECFCs transplant had also increased microvascular density. Modified neurological severity score and Morris water maze test indicated significant improvements in motor ability, spatial acquisition and reference memory in mice receiving ECFCs, compared to those receiving saline. These data demonstrate the beneficial effects of ECFC transplant on BBB integrity and angiogenesis in mice with TBI.
PMCID: PMC3868401  PMID: 23957220
endothelial colony-forming cells, endothelial progenitor cells; transplantation; traumatic brain injury
24.  Contraceptive Use and the Risk of Ectopic Pregnancy: A Multi-Center Case-Control Study 
PLoS ONE  2014;9(12):e115031.
To evaluate the association between the risk of ectopic pregnancy (EP) and the use of common contraceptives during the previous and current conception/menstrual cycle.
A multi-center case-control study was conducted in Shanghai. Women diagnosed with EP were recruited as the case group (n = 2,411). Women with intrauterine pregnancy (IUP) (n = 2,416) and non-pregnant women (n = 2,419) were matched as controls at a ratio of 1∶1. Information regarding the previous and current use of contraceptives was collected. Multivariate logistic regression analyses were performed to calculate odds ratios (ORs) and the corresponding 95% confidential intervals (CIs).
Previous use of intrauterine devices (IUDs) was associated with a slight risk of ectopic pregnancy (AOR1 = 1.87 [95% CI: 1.48–2.37]; AOR2 = 1.84 [1.49–2.27]), and the risk increased with the duration of previous use (P1 for trend <10−4, P2 for trend <10−4). The current use of most contraceptives reduced the risk of both unwanted IUP (condom: AOR = 0.04 [0.03–0.05]; withdrawal method: AOR = 0.10 [0.07–0.13]; calendar rhythm method: AOR = 0.54 [0.40–0.73]; oral contraceptive pills [OCPs]: AOR = 0.03 [0.02–0.08]; levonorgestrel emergency contraception [LNG-EC]: AOR = 0.22 [0.16–0.30]; IUDs: AOR = 0.01 [0.005–0.012]; tubal sterilization: AOR = 0.01 [0.001–0.022]) and unwanted EP (condom: AOR1 = 0.05 [0.04–0.06]; withdrawal method: AOR1 = 0.13 [0.09–0.19]; calendar rhythm method: AOR1 = 0.66 [0.48–0.91]; OCPs: AOR1 = 0.14 [0.07–0.26]; IUDs: AOR1 = 0.17 [0.13–0.22]; tubal sterilization: AOR1 = 0.04 [0.02–0.08]). However, when contraception failed and pregnancy occurred, current use of OCPs (AOR2 = 4.06 [1.64–10.07]), LNG-EC (AOR2 = 4.87 [3.88–6.10]), IUDs (AOR2 = 21.08 [13.44–33.07]), and tubal sterilization (AOR2 = 7.68 [1.69–34.80]) increased the risk of EP compared with the non-use of contraceptives.
Current use of most contraceptives reduce the risk of both IUP and EP. However, if the contraceptive method fails, the proportions of EP may be higher than those of non-users. In the case of contraceptive failure in the current cycle, EP cases should be differentiated according to current use of OCPs, LNG-EC, IUDs, and tubal sterilization. In addition, attention should be paid to women with previous long-term use of IUDs.
PMCID: PMC4262460  PMID: 25493939
25.  Risk factors and clinical features of ovarian pregnancy: a case–control study 
BMJ Open  2014;4(12):e006447.
To identify risk factors for ovarian pregnancy (OP) and compare clinical features between OP and tubal pregnancy (TP) patients.
Case–control study.
University hospital.
A case–control study was conducted from January 2005 to May 2014. Women diagnosed with OP were recruited as the case group (n=71), 145 women with TP and 146 with intrauterine pregnancy (IUP) were matched as controls at a ratio of 1:2:2. Women who refused interviews or provided incomplete information were excluded.
OP risk was lower than TP risk in women with serological evidence of Chlamydia trachomatis infection (adjusted OR1 0.17, 95% CI 0.06 to 0.52), previous adnexal surgery (adjusted OR1 0.25, 95% CI 0.07 to 0.95), and current levonorgestrel emergency contraceptive use (adjusted OR1 0.24, 95% CI 0.07 to 0.78). In vitro fertilisation-embryo transfer (IVF-ET) carried a higher risk of OP (adjusted OR1 12.18, 95% CI 2.23 to 66.58) than natural conception. When Controlled by IUP women, current users of intrauterine devices (IUDs) carried a higher risk of OP than non-users of any contraceptives (adjusted OR2 9.60, 95% CI 1.76 to 42.20). β-Human chorionic gonadotropin (hCG) levels on the day of surgery were higher in OP patients than in TP patients (p<0.01). Women with OP were less likely to initially present with vaginal bleeding than those with TP (p=0.02). Moreover, shock (p=0.02), rupture (p<0.01), haemoperitoneum (p<0.01) and emergency laparotomy (p<0.01) were more common in the OP group than in the TP group.
IVF-ET and IUD use may be risk factors for OP, and OP patients tend to have high β-hCG levels and a poor clinical outcome (shock, rupture, haemoperitoneum and need for emergency laparotomy). Our findings may contribute to the prevention and early diagnosis of OP.
PMCID: PMC4256641  PMID: 25472658

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