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1.  Analysis and Study on 47 Cases of Adverse Reactions of Chinese Medicine Injection 
Background
Along with efficacy, Chinese medicine is increasingly being known to people, Chinese medicine and its preparations are increasingly widespread in clinical use. People generally believe that Chinese medicine has few side effects and is safe, especially Chinese medicine injections. Due to the direct injection in blood, rapid onset and good efficacy, they are welcomed by people. However, with increased use, adverse reactions are increasing, even causing serious consequences. The objective is to learn about the characteristics and laws of the adverse effects of Chinese medicine injections, provide references for the clinical safe drug use, and reduce the incidence of adverse reactions.
Materials and Methods
The method was established to analyze the data of 47 cases of adverse reactions caused by Chinese medicine injections in our hospital from the year, 2009 to 2010.
Results
The organs / systems involved in the 47 cases of adverse reactions are primarily skin and its accessories and secondly systemic damage, involving a total of eight varieties of drugs.
Conclusion
The adverse reactions of Chinese medicine injections are mostly in Chinese patent drugs, which should be paid attention to, to find out the problems and laws, use the drugs rationally, and reduce the incidence of the adverse reactions.
PMCID: PMC4202647  PMID: 25435623
Chinese medicine; Injection; Adverse reactions
2.  Induction, Purification and Characterization of a Novel Manganese Peroxidase from Irpex lacteus CD2 and Its Application in the Decolorization of Different Types of Dye 
PLoS ONE  2014;9(11):e113282.
Manganese peroxidase (MnP) is the one of the important ligninolytic enzymes produced by lignin-degrading fungi which has the great application value in the field of environmental biotechnology. Searching for new MnP with stronger tolerance to metal ions and organic solvents is important for the maximization of potential of MnP in the biodegradation of recalcitrant xenobiotics. In this study, it was found that oxalic acid, veratryl alcohol and 2,6-Dimehoxyphenol could stimulate the synthesis of MnP in the white-rot fungus Irpex lacteus CD2. A novel manganese peroxidase named as CD2-MnP was purified and characterized from this fungus. CD2-MnP had a strong capability for tolerating different metal ions such as Ca2+, Cd2+, Co2+, Mg2+, Ni2+ and Zn2+ as well as organic solvents such as methanol, ethanol, DMSO, ethylene glycol, isopropyl alcohol, butanediol and glycerin. The different types of dyes including the azo dye (Remazol Brilliant Violet 5R, Direct Red 5B), anthraquinone dye (Remazol Brilliant Blue R), indigo dye (Indigo Carmine) and triphenylmethane dye (Methyl Green) as well as simulated textile wastewater could be efficiently decolorized by CD2-MnP. CD2-MnP also had a strong ability of decolorizing different dyes with the coexistence of metal ions and organic solvents. In summary, CD2-MnP from Irpex lacteus CD2 could effectively degrade a broad range of synthetic dyes and exhibit a great potential for environmental biotechnology.
doi:10.1371/journal.pone.0113282
PMCID: PMC4239052  PMID: 25412169
3.  Astragalus saponins modulates colon cancer development by regulating calpain-mediated glucose-regulated protein expression 
Background
Glucose-regulated proteins (GRP) are induced in the cancer microenvironment to promote tumor survival, metastasis and drug resistance. AST was obtained from the medicinal plant Astragalus membranaceus, which possesses anti-tumor and pro-apoptotic properties in colon cancer cells and tumor xenograft. The present study aimed to investigate the involvement of GRP in endoplasmic reticulum (ER) stress-mediated apoptosis during colon cancer development, with focus on the correlation between AST-evoked regulation of GRP and calpain activation.
Methods
The effects of AST on GRP and apoptotic activity were assessed in HCT 116 human colon adenocarcinoma cells. Calpain activity was examined by using a fluorescence assay kit. Immunofluorescence staining and immunoprecipitation were employed to determine the localization and association between calpains and GRP. GRP78 gene silencing was performed to confirm the importance of GRP in anticancer drug activities. The modulation of GRP and calpains was also studied in nude mice xenograft.
Results
ER stress-mediated apoptosis was induced by AST, as shown by elevation in both spliced XBP-1 and CHOP levels, with parallel up-regulation of GRP. The expression of XBP-1 and CHOP continued to increase after the peak level of GRP was attained at 24 h. Nevertheless, the initial increase in calpain activity as well as calpain I and II protein level was gradually declined at later stage of drug treatment. Besides, the induction of GRP was partly reversed by calpain inhibitors, with concurrent promotion of AST-mediated apoptosis. The knockdown of GRP78 by gene silencing resulted in higher sensitivity of colon cancer cells to AST-induced apoptosis and reduction of colony formation. The association between calpains and GRP78 had been confirmed by immunofluorescence staining and immunoprecipitation. Modulation of GRP and calpains by AST was similarly demonstrated in nude mice xenograft, leading to significant inhibition of tumor growth.
Conclusions
Our findings exemplify that calpains, in particular calpain II, play a permissive role in the modulation of GRP78 and consequent regulation of ER stress-induced apoptosis. Combination of calpain inhibitors and AST could exhibit a more pronounced pro-apoptotic effect. These results help to envisage a new therapeutic approach in colon cancer by targeting calpain and GRP.
Electronic supplementary material
The online version of this article (doi:10.1186/1472-6882-14-401) contains supplementary material, which is available to authorized users.
doi:10.1186/1472-6882-14-401
PMCID: PMC4210535  PMID: 25319833
GRP78; Calpain inhibitor; ER stress; Gene silencing; AST; Apoptosis; Metastasis; Tumor xenograft
4.  Azo Dye Biodecolorization Enhanced by Echinodontium taxodii Cultured with Lignin 
PLoS ONE  2014;9(10):e109786.
Lignocellulose facilitates the fungal oxidization of recalcitrant organic pollutants through the extracellular ligninolytic enzymes induced by lignin in wood or other plant tissues. However, available information on this phenomenon is insufficient. Free radical chain reactions during lignin metabolism are important in xenobiotic removal. Thus, the effect of lignin on azo dye decolorization in vivo by Echinodontium taxodii was evaluated. In the presence of lignin, optimum decolorization percentages for Remazol Brilliant Violet 5R, Direct Red 5B, Direct Black 38, and Direct Black 22 were 91.75% (control, 65.96%), 76.89% (control, 43.78%), 43.44% (control, 17.02%), and 44.75% (control, 12.16%), respectively, in the submerged cultures. Laccase was the most important enzyme during biodecolorization. Aside from the stimulating of laccase activity, lignin might be degraded by E. taxodii, and then these degraded low-molecular-weight metabolites could act as redox mediators promoting decolorization of azo dyes. The relationship between laccase and lignin degradation was investigated through decolorization tests in vitro with purified enzyme and dozens of aromatics, which can be derivatives of lignin and can function as laccase mediators or inducers. Dyes were decolorized at triple or even higher rates in certain laccase–aromatic systems at chemical concentrations as low as 10 µM.
doi:10.1371/journal.pone.0109786
PMCID: PMC4186836  PMID: 25285777
5.  Association between the Hypomethylation of Osteopontin and Integrin β3 Promoters and Vascular Smooth Muscle Cell Phenotype Switching in Great Saphenous Varicose Veins 
Lower extremity varicose veins are a common condition in vascular surgery and proliferation of vascular smooth muscle cells (VSMCs) in the intima is a significant pathological feature of varicosity. However, the pathogenesis of varicose veins is not fully understood. Osteopontin (OPN) could promote the migration and adhesion of VSMCs through the cell surface receptor integrin β3 and the cooperation of OPN and integrin β3 is involved in many vascular diseases. However, the role of OPN and integrin β3 in varicosity remains unclear. In the current study, we found that the methylation levels in the promoter regions of OPN and integrin β3 genes in the VSMCs of varicose veins are reduced and the protein expression of OPN and integrin β3 are increased, compared with normal veins. Furthermore, it was observed that VSMCs in the neointima of varicose veins were transformed into the synthetic phenotype. Collectively, hypomethylation of the promoter regions for OPN and integrin β3 genes may increase the expression of these genes in varicosity, which is closely related to VSMC phenotype switching. Hypomethylation of the promoter regions for OPN and integrin β3 genes may be a key factor in the pathogenesis of varicosity.
doi:10.3390/ijms151018747
PMCID: PMC4227244  PMID: 25329616
varicosity; vascular smooth muscle cells; phenotype switching; osteopontin; integrin β3; DNA methylation
6.  Let-7d suppresses growth, metastasis, and tumor macrophage infiltration in renal cell carcinoma by targeting COL3A1 and CCL7 
Molecular Cancer  2014;13(1):206.
Background
MicroRNAs are endogenous small noncoding RNAs that are functionally involved in numerous critical cellular processes including tumorigenesis. Data mining using a microRNA array database suggested that let-7d microRNA may be associated with renal cell carcinoma (RCC) malignant progression. Here, we performed further analyses to determine whether let-7d is functionally linked to RCC malignancy.
Methods
Quantitative real-time PCR was used to determine the level of mature let-7d in RCC clinical specimens and its correlation with clinicopathological data. Immunohistochemical staining was conducted to characterize the stroma of RCC. Let-7d overexpressing RCC cell lines combined with mouse models bearing cell-derived xenografts and patient-derived xenografts were used to assess the functional role of let-7d in vitro and in vivo.
Results
Downregulation of let-7d in clinical RCC samples was associated with advanced tumor grade and T stage and increased vascular invasion. An inverse relationship between let-7d expression and macrophage infiltration was found in clinical RCC samples. Functional studies indicated that ectopic expression of let-7d significantly inhibited RCC cell proliferation, migration, and peripheral blood monocyte (PBMC) recruitment in vitro, as well as tumor growth, metastasis, and tumor macrophage infiltration in vivo. In silico analysis and subsequent experimental validation confirmed collagen, type III, alpha 1 (COL3A1) and C-C subfamily chemokine member CCL7 as direct let-7d target genes. The addition of COL3A1 and CCL7 counteracted the inhibitory effects of let-7d on RCC cell proliferation, migration, and PBMC recruitment. The inhibition of let-7d increased cell proliferation, migration, and PBMC recruitment by the enhanced expression of COL3A1 and CCL7 genes in vitro. The mRNA levels of COL3A1 and CCL7 were inversely correlated with let-7d level in RCC clinical specimens.
Conclusions
These results suggest that let-7d may suppress RCC growth, metastasis, and tumor macrophage infiltration at least partially through targeting COL3A1 and CCL7.
Electronic supplementary material
The online version of this article (doi:10.1186/1476-4598-13-206) contains supplementary material, which is available to authorized users.
doi:10.1186/1476-4598-13-206
PMCID: PMC4168121  PMID: 25193015
Renal cell carcinoma; MicroRNA; Let-7
7.  Arabidopsis MRG domain proteins bridge two histone modifications to elevate expression of flowering genes 
Nucleic Acids Research  2014;42(17):10960-10974.
Trimethylation of lysine 36 of histone H3 (H3K36me3) is found to be associated with various transcription events. In Arabidopsis, the H3K36me3 level peaks in the first half of coding regions, which is in contrast to the 3′-end enrichment in animals. The MRG15 family proteins function as ‘reader’ proteins by binding to H3K36me3 to control alternative splicing or prevent spurious intragenic transcription in animals. Here, we demonstrate that two closely related Arabidopsis homologues (MRG1 and MRG2) are localised to the euchromatin and redundantly ensure the increased transcriptional levels of two flowering time genes with opposing functions, FLOWERING LOCUS C and FLOWERING LOCUS T (FT). MRG2 directly binds to the FT locus and elevates the expression in an H3K36me3-dependent manner. MRG1/2 binds to H3K36me3 with their chromodomain and interact with the histone H4-specific acetyltransferases (HAM1 and HAM2) to achieve a high expression level through active histone acetylation at the promoter and 5′ regions of target loci. Together, this study presents a mechanistic link between H3K36me3 and histone H4 acetylation. Our data also indicate that the biological functions of MRG1/2 have diversified from their animal homologues during evolution, yet they still maintain their conserved H3K36me3-binding molecular function.
doi:10.1093/nar/gku781
PMCID: PMC4176166  PMID: 25183522
8.  Nomogram Predicting Renal Insufficiency after Nephroureterectomy for Upper Tract Urothelial Carcinoma in the Chinese Population: Exclusion of Ineligible Candidates for Adjuvant Chemotherapy 
BioMed Research International  2014;2014:529186.
Objectives. To report the decline of renal function after radical nephroureterectomy (RNU) in upper tract urothelial carcinoma (UTUC) patients and to develop a nomogram to predict ineligibility for cisplatin-based adjuvant chemotherapy (AC). Methods. We retrospectively analyzed 606 consecutive Chinese UTUC patients treated by RNU from 2000 to 2010. We chose an eGFR of 60 and 45 ml/min/1.73 m2 as cut-offs for full-dose and reduced-dose AC eligibility. Results. Median eGFR for all patients before and after surgery was 64 and 49 ml/min/1.73 m2 (P < 0.001). The proportion of patients ineligible to receive full-dose and reduced-dose AC changed from 42% to 74% and from 20% to 38.1%. Older age (OR = 1.007), preoperative eGFR (OR = 0.993), absence of hydronephrosis (OR = 0.801), smaller tumor size (OR = 0.962), and tumor without multifocality (OR = 0.876) were predictive for ineligibility for full-dose AC. Preoperative eGFR (OR = 0.991), absence of hydronephrosis (OR = 0.881), tumor located in renal pelvis (OR = 1.164), and smaller tumor size (OR = 0.969) could predict ineligibility for reduced-dose AC. The c-index of the two models was 0.757 and 0.836. Postoperative renal function was not associated with worse survival. Conclusions. Older age, lower preoperative eGFR, smaller tumor size, tumor located in renal pelvis, and absence of hydronephrosis or multifocality were predictors of postoperative renal insufficiency.
doi:10.1155/2014/529186
PMCID: PMC4142385  PMID: 25180185
9.  Anomaly Detection Based on Local Nearest Neighbor Distance Descriptor in Crowded Scenes 
The Scientific World Journal  2014;2014:632575.
We propose a novel local nearest neighbor distance (LNND) descriptor for anomaly detection in crowded scenes. Comparing with the commonly used low-level feature descriptors in previous works, LNND descriptor has two major advantages. First, LNND descriptor efficiently incorporates spatial and temporal contextual information around the video event that is important for detecting anomalous interaction among multiple events, while most existing feature descriptors only contain the information of single event. Second, LNND descriptor is a compact representation and its dimensionality is typically much lower than the low-level feature descriptor. Therefore, not only the computation time and storage requirement can be accordingly saved by using LNND descriptor for the anomaly detection method with offline training fashion, but also the negative aspects caused by using high-dimensional feature descriptor can be avoided. We validate the effectiveness of LNND descriptor by conducting extensive experiments on different benchmark datasets. Experimental results show the promising performance of LNND-based method against the state-of-the-art methods. It is worthwhile to notice that the LNND-based approach requires less intermediate processing steps without any subsequent processing such as smoothing but achieves comparable event better performance.
doi:10.1155/2014/632575
PMCID: PMC4106157  PMID: 25105164
10.  Hypertensive stretch regulates endothelial exocytosis of Weibel-Palade bodies through VEGF receptor 2 signaling pathways 
Cell Research  2013;23(6):820-834.
Regulated endothelial exocytosis of Weibel-Palade bodies (WPBs), the first stage in leukocyte trafficking, plays a pivotal role in inflammation and injury. Acute mechanical stretch has been closely associated with vascular inflammation, although the precise mechanism is unknown. Here, we show that hypertensive stretch regulates the exocytosis of WPBs of endothelial cells (ECs) through VEGF receptor 2 (VEGFR2) signaling pathways. Stretch triggers a rapid release (within minutes) of von Willebrand factor and interleukin-8 from WPBs in cultured human ECs, promoting the interaction between leukocytes and ECs through the translocation of P-selectin to the cell membrane. We further show that hypertensive stretch significantly induces P-selectin translocation of intact ECs and enhances leukocyte adhesion both ex vivo and in vivo. Stretch-induced endothelial exocytosis is mediated via a VEGFR2/PLCγ1/calcium pathway. Interestingly, stretch also induces a negative feedback via a VEGFR2/Akt/nitric oxide pathway. Such dual effects are confirmed using pharmacological and genetic approaches in carotid artery segments, as well as in acute hypertensive mouse models. These studies reveal mechanical stretch as a potent agonist for endothelial exocytosis, which is modulated by VEGFR2 signaling. Thus, VEGFR2 signaling pathways may represent novel therapeutic targets in limiting hypertensive stretch-related inflammation.
doi:10.1038/cr.2013.56
PMCID: PMC3674392  PMID: 23609797
endothelial cells; stretch; VEGF receptor; exocytosis of Weibel-Palade bodies
11.  Preoperative Aspiration Culture for Preoperative Diagnosis of Infection in Total Hip or Knee Arthroplasty 
Journal of Clinical Microbiology  2013;51(11):3830-3834.
This meta-analysis evaluated preoperative aspiration culture for diagnosing prosthetic joint infection (PJI) in total hip arthroplasty (THA) and total knee arthroplasty (TKA). The pooled sensitivity and specificity were 0.72 (95% confidence interval, 0.65 to 0.78) and 0.95 (0.93 to 0.97), respectively. Subgroup analyses revealed nonsignificant worse diagnostic performance for THA than for TKA (sensitivity, 0.70 versus 0.78; specificity, 0.94 versus 0.96). Preoperative aspiration culture has moderate to high sensitivity and very high specificity for diagnosing PJI.
doi:10.1128/JCM.01467-13
PMCID: PMC3889774  PMID: 23946521
12.  Laparoscopic versus Open Total Gastrectomy for Gastric Cancer: An Updated Meta-Analysis 
PLoS ONE  2014;9(2):e88753.
Objective
To expand the current knowledge on the feasibility and safety of laparoscopic total gastrectomy (LTG) for gastric cancer in comparison with open total gastrectomy (OTG).
Background
Additional studies comparing laparoscopic versus open total gastric resection have been published, and it is necessary to update the meta-analysis of this subject.
Methods
Original articles compared LTG and OTG for gastric cancer, which published in English from January 1990 to July 2013 were searched in PubMed, Embase, and Web of Knowledge by two reviewers independently. Operative time, blood loss, harvested lymph nodes, proximal resection margin, analgesic medication, first flatus day, first oral intake, postoperative hospital stay, postoperative complications, hospital mortality, 5-year overall survival (OS) and disease-free survival (DFS) were compared using STATA version 10.1.
Results
17 studies were selected in this analysis, which included a total of 2313 patients (955 in LTG and 1358 in OTG). LTG showed longer operative time, less blood loss, fewer analgesic uses, earlier passage of flatus, quicker resumption of oral intake, earlier hospital discharge, and reduced postoperative morbidity. The number of harvested lymph nodes, proximal resection margin, hospital mortality, 5-year OS and DFS were similar.
Conclusion
LTG had the benefits of less blood loss, less postoperative pain, quicker bowel function recovery, shorter hospital stay and lower postoperative morbidity, at the price of longer operative time. There were no statistical differences in lymph node dissection, resection margin, hospital mortality, and long-term outcomes, which indicated the similar oncological safety with OTG. A positive trend was indicated towards LTG. So LTG can be performed as an alternative to OTG by the experienced surgeons in high-volume centers. Whereas, due to the relative small sample size of long-term outcomes and lack of randomized control trials, more studies are required.
doi:10.1371/journal.pone.0088753
PMCID: PMC3928285  PMID: 24558421
13.  Inhibition of HSP90 attenuates porcine reproductive and respiratory syndrome virus production in vitro 
Virology Journal  2014;11:17.
Background
Porcine reproductive and respiratory syndrome virus (PRRSV) infection leads to substantial economic losses to the swine industry worldwide. However, no effective countermeasures exist to combat this virus infection so far. The most common antiviral strategy relies on directly inhibiting viral proteins. However, this strategy invariably leads to the emergence of drug resistance due to the error-prone nature of viral ploymerase. Targeting cellular proteins required for viral infection for developing new generation of antivirals is gaining concern. Recently, heat shock protein 90 (HSP90) was found to be an important host factor for the replication of multiple viruses and the inhibition of HSP90 showed significant antiviral effects. It is thought that the inhibition of HSP90 could be a promising broad-range antiviral approach. However, the effects of HSP90 inhibition on PRRSV infection have not been evaluated. In the current research, we tried to inhibit HSP90 and test whether the inhibition affect PRRSV infection.
Methods
We inhibit the function of HSP90 with two inhibitors, geldanamycin (GA) and 17- allylamono-demethoxygeldanamycin (17-AAG), and down-regulated the expression of endogenous HSP90 with specific small-interfering RNAs (siRNAs). Cell viability was measured with alamarBlue. The protein level of viral N was determined by western blotting and indirect immunofluorescence (IFA). Besides, IFA was employed to examine the level of viral double-stranded RNA (dsRNA). The viral RNA copy number and the level of IFN-β mRNA were determined by quantitative real-time PCR (qRT-PCR).
Results
Our results indicated that both HSP90 inhibitors showed strong anti-PRRSV activity. They could reduce viral production by preventing the viral RNA synthesis. These inhibitory effects were not due to the activation of innate interferon response. In addition, we observed that individual knockdown targeting HSP90α or HSP90β did not show dramatic inhibitory effect. Combined knockdown of these two isoforms was required to reduce viral infection.
Conclusions
Our results shed light on the possibility of developing potential therapeutics targeting HSP90 against PRRSV infection.
doi:10.1186/1743-422X-11-17
PMCID: PMC3942275  PMID: 24490822
Porcine reproductive and respiratory syndrome virus; PRRSV; HSP90; Geldanamycin; 17-AAG; Antiviral
14.  Enhancing the Laccase Production and Laccase Gene Expression in the White-Rot Fungus Trametes velutina 5930 with Great Potential for Biotechnological Applications by Different Metal Ions and Aromatic Compounds 
PLoS ONE  2013;8(11):e79307.
Laccase is useful for various biotechnological and industrial applications. The white-rot fungus Trametes velutina 5930 and its laccase, isolated from the Shennongjia Nature Reserve in China by our laboratory, has great potential for practical application in environmental biotechnology. However, the original level of laccase produced by Trametes velutina 5930 was relatively low in the absence of any inducer. Therefore, in order to enhance the laccase production by Trametes velutina 5930 and make better use of this fungus in the field of environmental biotechnology, the regulation of laccase production and laccase gene expression in Trametes velutina 5930 were investigated in this study. Different metal ions such as Cu2+ and Fe2+ could stimulate the laccase synthesis and laccase gene transcription in Trametes velutina 5930. Some aromatic compounds structurally related to lignin, such as tannic acid, syringic acid, cinnamic acid, gallic acid and guaiacol, could also enhance the level of laccase activity and laccase gene transcription. We also found that there existed a positive synergistic effect of aromatic compound and metal ion on the laccase production and laccase gene transcription in Trametes velutina 5930. Taken together, our study may contribute to the improvement of laccase productivity by Trametes velutina 5930.
doi:10.1371/journal.pone.0079307
PMCID: PMC3823595  PMID: 24244475
15.  Natural Antioxidant-Isoliquiritigenin Ameliorates Contractile Dysfunction of Hypoxic Cardiomyocytes via AMPK Signaling Pathway 
Mediators of Inflammation  2013;2013:390890.
Isoliquiritigenin (ISL), a simple chalcone-type flavonoid, is derived from licorice compounds and is mainly present in foods, beverages, and tobacco. Reactive oxygen species (ROS) is a critical factor involved in modulating cardiac stress response signaling during ischemia and reperfusion. We hypothesize that ISL as a natural antioxidant may protect heart against ischemic injury via modulating cellular redox status and regulating cardioprotective signaling pathways. The fluorescent probe H2DCFDA was used to measure the level of intracellular ROS. The glucose uptake was determined by 2-deoxy-D-glucose-3H accumulation. The IonOptix System measured the contractile function of isolated cardiomyocytes. The results demonstrated that ISL treatment markedly ameliorated cardiomyocytes contractile dysfunction caused by hypoxia. ISL significantly stimulated cardioprotective signaling, AMP-activated protein kinase (AMPK), and extracellular signal-regulated kinase (ERK) signaling pathways. The ROS fluorescent probe H2DCFDA determination indicated that ISL significantly reduced cardiac ROS level during hypoxia/reoxygenation. Moreover, ISL reduced the mitochondrial potential (Δψ) of isolated mouse cardiomyocytes. Taken together, ISL as a natural antioxidant demonstrated the cardioprotection against ischemic injury that may attribute to the activation of AMPK and ERK signaling pathways and balance of cellular redox status.
doi:10.1155/2013/390890
PMCID: PMC3791876  PMID: 24163504
16.  The effect of Shenmai injection on the proliferation of Rat airway smooth muscle cells in asthma and underlying mechanism 
Background
Over-proliferation of airway smooth muscle cell (ASMC) is one of the important contributors to airway remodeling in asthma. The aim of this study was to investigate the effect of Shenmai injection (SMI) on the proliferation of the rat ASMC in asthma.
Methods
Rats were randomly divided into three groups: the control group, the asthma group, and the SMI treatment group. Reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry staining were used to detect the mRNA and protein expression of transient receptor potential vanilloid 1 (TRPV1) and proliferating cell nuclear antigen (PCNA) in rat ASMC respectively. Intracellular Ca2+ concentration ( [Ca2+]i ) in rat ASMC were measured with Fluo-3/AM by confocal microscopy. The proliferation was detected by MTT assay.
Results
Compared with the control group, the asthma group showed an increased expression of TRPV1 and [Ca2+]i in rat ASMC. The expression of PCNA and absorbance of MTT assay in asthma rat ASMC was also significantly increased. SMI could significantly decrease the expression of TRPV1 channel and [Ca2+]i in the asthmatic rat ASMC. Furthermore, the expression of PCNA and absorbance of MTT assay in asthmatic rat ASMC was significantly reduced after SMI treatment.
Conclusions
SMI may prevent asthma-induced ASMC over-proliferation probably by inhibiting the expression of TRPV1 channel, which regulates the intracellular calcium concentration.
doi:10.1186/1472-6882-13-221
PMCID: PMC3849921  PMID: 24010863
Shenmai injection (SMI); Transient receptor potential vanilloid 1 (TRPV1); Airway smooth muscle cells (ASMC); Intracellular calcium; Airway remodeling
17.  Expression of the Laccase Gene from a White Rot Fungus in Pichia pastoris Can Enhance the Resistance of This Yeast to H2O2-Mediated Oxidative Stress by Stimulating the Glutathione-Based Antioxidative System 
Applied and Environmental Microbiology  2012;78(16):5845-5854.
Laccase is a copper-containing polyphenol oxidase that has great potential in industrial and biotechnological applications. Previous research has suggested that fungal laccase may be involved in the defense against oxidative stress, but there is little direct evidence supporting this hypothesis, and the mechanism by which laccase protects cells from oxidative stress also remains unclear. Here, we report that the expression of the laccase gene from white rot fungus in Pichia pastoris can significantly enhance the resistance of yeast to H2O2-mediated oxidative stress. The expression of laccase in yeast was found to confer a strong ability to scavenge intracellular H2O2 and to protect cells from lipid oxidative damage. The mechanism by which laccase gene expression increases resistance to oxidative stress was then investigated further. We found that laccase gene expression in Pichia pastoris could increase the level of glutathione-based antioxidative activity, including the intracellular glutathione levels and the enzymatic activity of glutathione peroxidase, glutathione reductase, and γ-glutamylcysteine synthetase. The transcription of the laccase gene in Pichia pastoris was found to be enhanced by the oxidative stress caused by exogenous H2O2. The stimulation of laccase gene expression in response to exogenous H2O2 stress further contributed to the transcriptional induction of the genes involved in the glutathione-dependent antioxidative system, including PpYAP1, PpGPX1, PpPMP20, PpGLR1, and PpGSH1. Taken together, these results suggest that the expression of the laccase gene in Pichia pastoris can enhance the resistance of yeast to H2O2-mediated oxidative stress by stimulating the glutathione-based antioxidative system to protect the cell from oxidative damage.
doi:10.1128/AEM.00218-12
PMCID: PMC3406150  PMID: 22706050
18.  Widespread splicing changes in human brain development and aging 
Human brain transcriptome analysis revealed widespread age-related splicing changes in the prefrontal cortex and cerebellum. While most of the splicing changes take place in development, approximately one-third of them extends into aging.
More than one-third of genes expressed in the human brain change splicing with age.Approximately 30% of observed splicing changes occur in aging.Age-related splicing patterns are largely conserved between the human and macaque brains.High frequency of intron retention events suggests the role of nonsense-mediated decay in age-related gene expression regulation.
While splicing differences between tissues, sexes and species are well documented, little is known about the extent and the nature of splicing changes that take place during human or mammalian development and aging. Here, using high-throughput transcriptome sequencing, we have characterized splicing changes that take place during whole human lifespan in two brain regions: prefrontal cortex and cerebellum. Identified changes were confirmed using independent human and rhesus macaque RNA-seq data sets, exon arrays and PCR, and were detected at the protein level using mass spectrometry. Splicing changes across lifespan were abundant in both of the brain regions studied, affecting more than a third of the genes expressed in the human brain. Approximately 15% of these changes differed between the two brain regions. Across lifespan, splicing changes followed discrete patterns that could be linked to neural functions, and associated with the expression profiles of the corresponding splicing factors. More than 60% of all splicing changes represented a single splicing pattern reflecting preferential inclusion of gene segments potentially targeting transcripts for nonsense-mediated decay in infants and elderly.
doi:10.1038/msb.2012.67
PMCID: PMC3564255  PMID: 23340839
alternative splicing; brain; development; human; RNA-seq
19.  Spreading of Heterochromatin Is Limited to Specific Families of Maize Retrotransposons 
PLoS Genetics  2012;8(12):e1003127.
Transposable elements (TEs) have the potential to act as controlling elements to influence the expression of genes and are often subject to heterochromatic silencing. The current paradigm suggests that heterochromatic silencing can spread beyond the borders of TEs and influence the chromatin state of neighboring low-copy sequences. This would allow TEs to condition obligatory or facilitated epialleles and act as controlling elements. The maize genome contains numerous families of class I TEs (retrotransposons) that are present in moderate to high copy numbers, and many are found in regions near genes, which provides an opportunity to test whether the spreading of heterochromatin from retrotransposons is prevalent. We have investigated the extent of heterochromatin spreading into DNA flanking each family of retrotransposons by profiling DNA methylation and di-methylation of lysine 9 of histone 3 (H3K9me2) in low-copy regions of the maize genome. The effects of different retrotransposon families on local chromatin are highly variable. Some retrotransposon families exhibit enrichment of heterochromatic marks within 800–1,200 base pairs of insertion sites, while other families exhibit very little evidence for the spreading of heterochromatic marks. The analysis of chromatin state in genotypes that lack specific insertions suggests that the heterochromatin in low-copy DNA flanking retrotransposons often results from the spreading of silencing marks rather than insertion-site preferences. Genes located near TEs that exhibit spreading of heterochromatin tend to be expressed at lower levels than other genes. Our findings suggest that a subset of retrotransposon families may act as controlling elements influencing neighboring sequences, while the majority of retrotransposons have little effect on flanking sequences.
Author Summary
Transposable elements comprise a substantial portion of many eukaryotic genomes. These mobile fragments of DNA can directly mutate genes through insertions into coding regions but may also affect the gene regulation through nearby insertions. There is evidence that the majority of transposable elements are epigenetically silenced, and in some cases this silencing may spread to neighboring sequences. This spreading of heterochromatin could create a significant fitness tradeoff between transposon silencing and gene expression. The maize genome has a complex organization with many genes flanked by retrotransposons, providing an opportunity to study the interaction of retrotransposons and genes. To survey the prevalence of heterochromatin spreading associated with different retrotransposon families, we profiled the spread of heterochromatin into nearby low copy sequences for 150 high copy retrotransposon families. While many retrotransposons exhibit little to no spreading of heterochromatin, there are some retrotransposon families that do exhibit spreading. Genes located near retrotransposons that spread heterochromatin have lower expression levels. The families of retrotransposons that spread heterochromatin marks to nearby low-copy sequences may have increased fitness costs for the host genome due to their suppression of genes located near insertions.
doi:10.1371/journal.pgen.1003127
PMCID: PMC3521669  PMID: 23271981
20.  Bone Marrow Vaccination: A Novel Approach to Enhance Antigen Specific Antitumor Immunity 
Vaccine  2011;29(47):8599-8605.
Bone marrow (BM) serves as a reservoir for a unique population of memory T cells with strong effector properties that make them ideal target for cancer immunotherapy strategies. However, direct vaccination and priming of T cells within the BM of the host has never been investigated. This study evaluates the specific immune response induced via a new method of direct intra-bone marrow (IBM) vaccinations in an animal model of human papillomavirus-associated cancer. We found that IBM vaccinations with the class I HPV-16 E7 epitope induce large numbers of activated, IFN-γ-producing E7-specific lymphocytes in the BM. In prophylactic tumor challenge experiments, direct intra-BM vaccination was found to be protective against tumor formation for 80% of the mice. In the therapeutic setting, IBM vaccination induced tumor regression in 3 of 10 vaccinated mice and delayed tumor growth in the remaining animals. Finally, adoptive transfer of BM cells from IBM vaccinated mice to naïve animals conferred complete protection against tumor growth. These data demonstrate the capacity of direct IBM vaccination to induce potent antigen-specific immunity resulting in protection from tumor growth in an animal model. Specifically targeting BM T cells with vaccines may improve responses to cancer immunotherapy and offer important clinical advantages, especially in the setting of bone marrow malignancies.
doi:10.1016/j.vaccine.2011.09.022
PMCID: PMC3200474  PMID: 21951877
Vaccination; Bone Marrow; memory T cells; HPV
21.  Interleukin-7 inhibits tumor-induced CD27−CD28− suppressor T-cells: implications for cancer immunotherapy 
Purpose
We have previously reported that many types of tumors are able to induce changes in human T cells that lead to the acquisition of suppressive function as well as phenotypic alterations resembling those found in senescent T cells. In the present study we find a role for IL-7 in protecting T cells from these changes, and further define involved signaling pathways.
Experimental Design
We evaluated the ability of IL-7 treatment to prevent the gain of suppressive function and phenotypic alterations in human T cells after a short co-culture with tumor cells in vitro. We then used inhibitors of components of the PI3K/AKT pathway and siRNA knock-down of Mcl-1 and Bim to evaluate the role of these signaling pathways in IL-7 protection.
Results
We found that IL-7 inhibits CD27/CD28 loss and maintains proliferative capacity, IL-2 production, and reduced suppressive function. IL-7’s protective ability depended on activation of the PI3K/AKT pathway, which inhibited activation of glycogen synthase kinase 3β (GSK3β) which subsequently prevented the phosphorylation and loss of Mcl-1. We further demonstrated a key role for Mcl-1 in that its knock-down or inhibition abrogated the effects of IL-7. Additionally, knock-down of the Mcl-1 binding partner and pro-apoptotic protein Bim protected T cells from these dysfunctional alterations.
Conclusion
These observations confirm the role for Bcl-2 family members in cytokine signaling, and suggest that IL-7 treatment in combination with other immunotherapies could lead to new clinical strategies to maintain normal T cell function and reduce tumor-induced generation of dysfunctional and suppressor T cells.
doi:10.1158/1078-0432.CCR-10-3328
PMCID: PMC3149850  PMID: 21712448
Interleukin-7; tumor immunology; immunosuppression; Mcl-1; immunotherapy
22.  MicroRNA-Driven Developmental Remodeling in the Brain Distinguishes Humans from Other Primates 
PLoS Biology  2011;9(12):e1001214.
Comparison of human, chimpanzee, and macaque brain transcriptomes reveals a significant developmental remodeling in the human prefrontal cortex, potentially shaped by microRNA.
While multiple studies have reported the accelerated evolution of brain gene expression in the human lineage, the mechanisms underlying such changes are unknown. Here, we address this issue from a developmental perspective, by analyzing mRNA and microRNA (miRNA) expression in two brain regions within macaques, chimpanzees, and humans throughout their lifespan. We find that constitutive gene expression divergence (species differences independent of age) is comparable between humans and chimpanzees. However, humans display a 3–5 times faster evolutionary rate in divergence of developmental patterns, compared to chimpanzees. Such accelerated evolution of human brain developmental patterns (i) cannot be explained by life-history changes among species, (ii) is twice as pronounced in the prefrontal cortex than the cerebellum, (iii) preferentially affects neuron-related genes, and (iv) unlike constitutive divergence does not depend on cis-regulatory changes, but might be driven by human-specific changes in expression of trans-acting regulators. We show that developmental profiles of miRNAs, as well as their target genes, show the fastest rates of human-specific evolutionary change, and using a combination of computational and experimental methods, we identify miR-92a, miR-454, and miR-320b as possible regulators of human-specific neural development. Our results suggest that different mechanisms underlie adaptive and neutral transcriptome divergence, and that changes in the expression of a few key regulators may have been a major driving force behind rapid evolution of the human brain.
Author Summary
Species evolution is often depicted as a slow and continuous process punctuated by rapid changes. One example of the latter is the evolution of human cognition–emergence of an exceedingly complex phenotype within a few million years. What genetic mechanisms might have driven this process? Nearly 40 years ago, it was proposed that human-specific gene expression changes, rather than changes in protein sequence, might underlie human cognitive evolution. Here we compare gene expression throughout postnatal brain development in humans, chimpanzees, and macaques. We find that simple changes in gene expression levels, plausibly driven by mutations in cis-regulatory elements, accumulate at similar rates in all three evolutionary lineages. What sharply distinguishes humans from other species is change in the timing and shape of developmental expression patterns. This is particularly pronounced in the prefrontal cortex, where 4-fold more genes show more human-specific developmental changes than chimpanzee-specific ones. Notably, our results indicate that this massive developmental remodeling of the human cortex, which affects hundreds of genes, might be driven by expression changes of only a few key regulators, such as microRNAs. Genes affected by this remodeling are preferentially associated with neural activity, thereby suggesting a link to the evolution of human cognition.
doi:10.1371/journal.pbio.1001214
PMCID: PMC3232219  PMID: 22162950
23.  MicroRNA Expression and Regulation in Human, Chimpanzee, and Macaque Brains 
PLoS Genetics  2011;7(10):e1002327.
Among other factors, changes in gene expression on the human evolutionary lineage have been suggested to play an important role in the establishment of human-specific phenotypes. However, the molecular mechanisms underlying these expression changes are largely unknown. Here, we have explored the role of microRNA (miRNA) in the regulation of gene expression divergence among adult humans, chimpanzees, and rhesus macaques, in two brain regions: prefrontal cortex and cerebellum. Using a combination of high-throughput sequencing, miRNA microarrays, and Q-PCR, we have shown that up to 11% of the 325 expressed miRNA diverged significantly between humans and chimpanzees and up to 31% between humans and macaques. Measuring mRNA and protein expression in human and chimpanzee brains, we found a significant inverse relationship between the miRNA and the target genes expression divergence, explaining 2%–4% of mRNA and 4%–6% of protein expression differences. Notably, miRNA showing human-specific expression localize in neurons and target genes that are involved in neural functions. Enrichment in neural functions, as well as miRNA–driven regulation on the human evolutionary lineage, was further confirmed by experimental validation of predicted miRNA targets in two neuroblastoma cell lines. Finally, we identified a signature of positive selection in the upstream region of one of the five miRNA with human-specific expression, miR-34c-5p. This suggests that miR-34c-5p expression change took place after the split of the human and the Neanderthal lineages and had adaptive significance. Taken together these results indicate that changes in miRNA expression might have contributed to evolution of human cognitive functions.
Author Summary
Humans are remarkably similar to apes and monkeys on the genome sequence level but remain remarkably distinct with respect to cognitive abilities. How could human cognition evolve within such a short evolutionary time? Among many hypotheses, evolution in expression of a few key regulators affecting hundreds of their target genes was proposed as one possible solution. Here, we tested this notion by studying expression divergence of a specific type of regulatory RNA, microRNA (miRNA), and its effect on gene expression profiles in brains of humans, chimpanzees, and rhesus macaques. Our results indicate that changes in miRNA expression have played a considerable role in the establishment of gene expression divergence between human brains and brains of non-human primates at both mRNA and protein expression levels. Furthermore, we find indications that some of the human-specific gene expression profiles caused by miRNA expression divergence might be associated with evolution of human-specific functions.
doi:10.1371/journal.pgen.1002327
PMCID: PMC3192836  PMID: 22022286
24.  The promoting effect of byproducts from Irpex lacteus on subsequent enzymatic hydrolysis of bio-pretreated cornstalks 
Background
Irpex lacteus, a versatile lignin-degrading fungus with various extracellular enzymes, has been widely used for biological pretreatment. However, most studies have focused on the change of substrate structure after biological pretreatment, and the effect of these changes on the enzymatic hydrolysis, but the effect of byproducts from biological pretreatment process on subsequent enzymatic hydrolysis is not well understood.
Methods
We developed a biological pretreatment process with I. lacteus that can produce stimulatory byproducts that enhance the enzymatic hydrolysis of cornstalks.
Results
The maximum hydrolysis yield of glucan (82%) was obtained after pretreatment for 28 days. The maximum reducing sugar yield decreased from 313.5 to 200.1 mg/g raw cornstalks after water-soluble byproducts of biological pretreatment were removed from pretreated cornstalks. The effect of byproducts on enzymatic hydrolysis was also investigated. We found that the hydrolysis efficiency of commercial cellulase preparation on cornstalks could be improved by water extracts from bio-pretreated cornstalks with hydrolytic enzyme activity and iron-reducing activity.
Conclusion
The key finding suggested that byproducts from biological pretreatment play important roles in enhancing downstream hydrolysis, which might be attributable to hydrolytic enzymes and iron-reducing compounds produced by I. lacteus.
doi:10.1186/1754-6834-4-37
PMCID: PMC3238224  PMID: 21985037
25.  High-throughput approaches for plant epigenomic studies 
Current opinion in plant biology  2011;14(2):130-136.
In plant cells, DNA is packaged into chromatin by wrapping around histone octamers. Pathways that lead to cytosine DNA methylation, posttranslational histone modifications and certain components of the RNA interfering (RNAi) pathway are critically important in modulating chromatin structure, thereby affecting many molecular processes that take place in a cell. Recent advances in microarray and high-throughput sequencing technologies have made it possible to study these pathways on a genome-wide scale. Results from such epigenomic studies are broadening our understanding of plant genomes and are also providing important clues regarding the mechanisms and functions of these pathways that can be further tested using genetic and biochemical approaches. This review focuses on the high-throughput approaches that have been successfully applied in plant epigenomic studies.
doi:10.1016/j.pbi.2011.03.010
PMCID: PMC3112054  PMID: 21470901

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