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1.  Effect of Bacillus amyloliquefaciens-based Direct-fed Microbial on Performance, Nutrient Utilization, Intestinal Morphology and Cecal Microflora in Broiler Chickens 
The present study was conducted to evaluate the effect of the dietary supplementation of Bacillus amyloliquefaciens-based direct-fed microbial (DFM) on growth performance, nutrient utilization, intestinal morphology and cecal microflora in broiler chickens. A total of two hundred and eighty eight 1-d-old Arbor Acres male broilers were randomly allocated to one of four experimental treatments in a completely randomized design. Each treatment was fed to eight replicate cages, with nine birds per cage. Dietary treatments were composed of an antibiotic-free basal diet (control), and the basal diet supplemented with either 15 mg/kg of virginiamycin as antibiotic growth promoter (AGP), 30 mg/kg of Bacillus amyloliquefaciens-based DFM (DFM 30) or 60 mg/kg of Bacillus amyloliquefaciens-based DFM (DFM 60). Experimental diets were fed in two phases: starter (d 1 to 21) and finisher (d 22 to 42). Growth performance, nutrient utilization, morphological parameters of the small intestine and cecal microbial populations were measured at the end of the starter (d 21) and finisher (d 42) phases. During the starter phase, DFM and virginiamycin supplementation improved the feed conversion ratio (FCR; p<0.01) compared with the control group. For the finisher phase and the overall experiment (d 1 to 42) broilers fed diets with the DFM had better body weight gain (BWG) and FCR than that of control (p<0.05). Supplementation of virginiamycin and DFM significantly increased the total tract apparent digestibility of crude protein (CP), dry matter (DM) and gross energy during both starter and finisher phases (p<0.05) compared with the control group. On d 21, villus height, crypt depth and villus height to crypt depth ratio of duodenum, jejunum, and ileum were significantly increased for the birds fed with the DFM diets as compared with the control group (p<0.05). The DFM 30, DFM 60, and AGP groups decreased the Escherichia coli population in cecum at d 21 and d 42 compared with control group (p<0.01). In addition, the population of Lactobacillus was increased in DFM 30 and DFM 60 groups as compared with control and AGP groups (p<0.01). It can be concluded that Bacillus amyloliquefaciens-based DFM could be an alternative to the use of AGPs in broilers diets based on plant protein.
PMCID: PMC4283169  PMID: 25557820
Bacillus amyloliquefaciens; Broilers; Cecal Microflora; Intestinal Morphology; Nutrient Utilization; Performance
2.  De Novo Assembly and Transcriptome Analysis of the Mediterranean Fruit Fly Ceratitis capitata Early Embryos 
PLoS ONE  2014;9(12):e114191.
The agricultural pest Ceratitis capitata, also known as the Mediterranean fruit fly or Medfly, belongs to the Tephritidae family, which includes a large number of other damaging pest species. The Medfly has been the first non-drosophilid fly species which has been genetically transformed paving the way for designing genetic-based pest control strategies. Furthermore, it is an experimentally tractable model, in which transient and transgene-mediated RNAi have been successfully used. We applied Illumina sequencing to total RNA preparations of 8–10 hours old embryos of C. capitata, This developmental window corresponds to the blastoderm cellularization stage. In summary, we assembled 42,614 transcripts which cluster in 26,319 unique transcripts of which 11,045 correspond to protein coding genes; we identified several hundreds of long ncRNAs; we found an enrichment of transcripts encoding RNA binding proteins among the highly expressed transcripts, such as CcTRA-2, known to be necessary to establish and, most likely, to maintain female sex of C. capitata. Our study is the first de novo assembly performed for Ceratitis capitata based on Illumina NGS technology during embryogenesis and it adds novel data to the previously published C. capitata EST databases. We expect that it will be useful for a variety of applications such as gene cloning and phylogenetic analyses, as well as to advance genetic research and biotechnological applications in the Medfly and other related Tephritidae.
PMCID: PMC4256415  PMID: 25474564
3.  Estradiol improves cardiovascular function through up-regulation of SOD2 on vascular wall 
Redox Biology  2014;3:88-99.
Epidemiological studies have shown that estrogens have protective effects in cardiovascular diseases, even though the results from human clinical trials remain controversial, while most of the animal experiments confirmed this effect, but the detailed mechanism remains unclear. In this study, we found that estradiol (E2) treatment significantly increases the expression of mitochondrial superoxide dismutase (SOD2) in mice and in vitro in human aorta endothelial cells. Further investigation shows that E2 up-regulates SOD2 through tethering of estrogen receptor (ER) to Sp1 and the increased binding of Sp1 to GC-box on the SOD2 promoter, where ERα responses E2-mediated gene activation, and ERβ maintains basal gene expression level. The E2/ER-mediated SOD2 up-regulation results in minimized ROS generation, which highly favors healthy cardiovascular function. Gene therapy through lentivirus-carried endothelium-specific delivery to the vascular wall in high-fat diet (HFT) mice shows that the SOD2 expression in endothelial cells normalizes E2 deficiency-induced ROS generation with ameliorated mitochondrial dysfunction and vascular damage, while SOD2 knockdown worsens the problem despite the presence of E2, indicating that E2-induced SOD2 expression plays an important vasculoprotective role. To our knowledge, this is the first report for the mechanism by which E2 improves cardiovascular function through up-regulation of SOD2 in endothelial cells. In turn, this suggests a novel gene therapy through lentivirus-carried gene delivery to vascular wall for E2 deficiency-induced cardiovascular damage in postmenopausal women.
PMCID: PMC4297935  PMID: 25462070
Endothelium; Estrogen receptor; Gene therapy; Mitochondrial function; Reactive oxygen species
4.  Destabilization of Heterologous Proteins Mediated by the GSK3β Phosphorylation Domain of the β-Catenin Protein 
Background and Aims
Wnt/β-catenin signaling plays important roles in development and cellular processes. The hallmark of canonical Wnt signaling activation is the stabilization of β-catenin protein in cytoplasm and/or nucleus. The stability of β-catenin is the key to its biological functions and is controlled by the phosphorylation of its amino-terminal degradation domain. Aberrant activation of β-catenin signaling has been implicated in the development of human cancers. It has been recently suggested that GSK3β may play an essential role in regulating global protein turnover. Here, we investigate if the GSK3β phosphorylation site-containing degradation domain of β-catenin is sufficient to destabilize heterologous proteins.
Methods and Results
We engineer chimeric proteins by fusing β-catenin degradation domain at the N- and/or C-termini of the enhanced green fluorescent protein (eGFP). In both transient and stable expression experiments, the chimeric GFP proteins exhibit a significantly decreased stability, which can be effectively antagonized by lithium and Wnt1. An activating mutation in the destruction domain significantly stabilizes the fusion protein. Furthermore, GSK3 inhibitor SB-216763 effectively increases the GFP signal of the fusion protein. Conversely, the inhibition of Wnt signaling with tankyrase inhibitor XAV939 results in a decrease in GFP signal of the fusion proteins, while these small molecules have no significant effects on the mutant destruction domain-GFP fusion protein.
Our findings strongly suggest that the β-catenin degradation domain may be sufficient to destabilize heterologous proteins in Wnt signaling-dependent manner. It is conceivable that the chimeric GFP proteins may be used as a functional reporter to measure the dynamic status of β-catenin signaling, and to identify potential anticancer drugs that target β-catenin signaling.
PMCID: PMC4064945  PMID: 24335169
β-catenin; Wnt signal; Human cancer; Protein degradation; Green fluorescent protein; Destabilized GFP; Chimeric proteins
5.  miR-193a-3p regulates the multi-drug resistance of bladder cancer by targeting the LOXL4 gene and the Oxidative Stress pathway 
Molecular Cancer  2014;13(1):234.
Chemoresistance is a major obstacle to the curative cancer chemotherapy and presents one of the most formidable challenges in both research and management of cancer.
From the detailed studies of a multi-chemosensitive (5637) versus a chemoresistant (H-bc) bladder cancer cell lines, we showed that miR-193a-3p [GenBank: NR_029710.1] promotes the multi-chemoresistance of bladder cancer cells. We further demonstrated that lysyl oxidase-like 4 (LOXL4) gene [GenBank: NM_032211.6] is a direct target of miR-193a-3p and executes the former’s impact on bladder cancer chemoresistance. The Oxidative Stress pathway activity is drastically affected by a forced reversal of miR-193a-3p or LOXL4 levels in cell and may act at the downstream of LOXL4 gene to relay the miR-193a-3p’s impact on the multi-chemoresistance in both cultured cells and the tumor xenografts in nude mice.
In addition to a new mechanistic insight, our results provide a set of the essential genes in this newly identified miR-193a-3p/LOXL4/Oxidative Stress axis as the diagnostic targets for a guided anti-bladder cancer chemotherapy.
Electronic supplementary material
The online version of this article (doi:10.1186/1476-4598-13-234) contains supplementary material, which is available to authorized users.
PMCID: PMC4200202  PMID: 25311867
LOXL4; miR-193a-3p; Chemoresistance; Bladder cancer; Oxidative stress pathway
6.  High-frequency aberrantly methylated targets in pancreatic adenocarcinoma identified via global DNA methylation analysis using methylCap-seq 
Clinical Epigenetics  2014;6(1):18.
Extensive reprogramming and dysregulation of DNA methylation is an important characteristic of pancreatic cancer (PC). Our study aimed to characterize the genomic methylation patterns in various genomic contexts of PC. The methyl capture sequencing (methylCap-seq) method was used to map differently methylated regions (DMRs) in pooled samples from ten PC tissues and ten adjacent non-tumor (PN) tissues. A selection of DMRs was validated in an independent set of PC and PN samples using methylation-specific PCR (MSP), bisulfite sequencing PCR (BSP), and methylation sensitive restriction enzyme-based qPCR (MSRE-qPCR). The mRNA and expressed sequence tag (EST) expression of the corresponding genes was investigated using RT-qPCR.
A total of 1,131 PC-specific and 727 PN-specific hypermethylated DMRs were identified in association with CpG islands (CGIs), including gene-associated CGIs and orphan CGIs; 2,955 PC-specific and 2,386 PN-specific hypermethylated DMRs were associated with gene promoters, including promoters containing or lacking CGIs. Moreover, 1,744 PC-specific and 1,488 PN-specific hypermethylated DMRs were found to be associated with CGIs or CGI shores. These results suggested that aberrant hypermethylation in PC typically occurs in regions surrounding the transcription start site (TSS). The BSP, MSP, MSRE-qPCR, and RT-qPCR data indicated that the aberrant DNA methylation in PC tissue and in PC cell lines was associated with gene (or corresponding EST) expression.
Our study characterized the genome-wide DNA methylation patterns in PC and identified DMRs that were distributed among various genomic contexts that might influence the expression of corresponding genes or transcripts to promote PC. These DMRs might serve as diagnostic biomarkers or therapeutic targets for PC.
PMCID: PMC4177372  PMID: 25276247
CGI shore; DNA methylation; genome-wide; methyl capture sequencing; orphan CGI; pancreatic adenocarcinoma
7.  Bacterial Communities in the Gut and Reproductive Organs of Bactrocera minax (Diptera: Tephritidae) Based on 454 Pyrosequencing 
PLoS ONE  2014;9(9):e106988.
The citrus fruit fly Bactrocera minax is associated with diverse bacterial communities. We used a 454 pyrosequencing technology to study in depth the microbial communities associated with gut and reproductive organs of Bactrocera minax. Our dataset consisted of 100,749 reads with an average length of 400 bp. The saturated rarefaction curves and species richness indices indicate that the sampling was comprehensive. We found highly diverse bacterial communities, with individual sample containing approximately 361 microbial operational taxonomic units (OTUs). A total of 17 bacterial phyla were obtained from the flies. A phylogenetic analysis of 16S rDNA revealed that Proteobacteria was dominant in all samples (75%–95%). Actinobacteria and Firmicutes were also commonly found in the total clones. Klebsiella, Citrobacter, Enterobacter, and Serratia were the major genera. However, bacterial diversity (Chao1, Shannon and Simpson indices) and community structure (PCA analysis) varied across samples. Female ovary has the most diverse bacteria, followed by male testis, and the bacteria diversity of reproductive organs is richer than that of the gut. The observed variation can be caused by sex and tissue, possibly to meet the host's physiological demands.
PMCID: PMC4162550  PMID: 25215866
8.  T-type Ca2+ channels regulate the exit of cardiac myocytes from the cell cycle after birth 
T-type Ca2+ channels (TTCCs) are expressed in the fetal heart and then disappear from ventricular myocytes after birth. The hypothesis examined in this study was the α1G TTCCs' influence in myocyte maturation and their rapid withdrawal from the cell cycle after birth.
Cardiac myocytes were isolated from neonatal and adult wild type (WT), α1G−/− and α1G over expressing (α1GDT) mice. Bromodeoxyuridine (BrdU) uptake, myocyte nucleation, cell cycle analysis, and T-type Ca2+ currents were measured.
All myocytes were mono-nucleated at birth and 35% of WT myocytes expressed functional TTCCs. Very few neonatal myocytes had functional TTCCs in α1G−/− hearts. By the end of the first week after birth no WT or α1G−/− had functional TTCCs. During the first week after birth about 25% of WT myocytes were BrdU+ and became bi-nucleated. Significantly fewer α1G−/− myocytes became bi-nucleated and fewer of these myocytes were BrdU+. Neonatal α1G−/− myocytes were also smaller than WT. Adult WT and α1G−/− hearts were similar in size, but α1G−/− myocytes were smaller and a greater % were mono-nucleated. α1G over expressing hearts were smaller than WT but their myocytes were larger.
The studies performed show that loss of functional TTCCs is associated with bi-nucleation and myocyte withdrawal from the cell cycle. Loss of α1G TTCCs slowed the transition from mono- to bi-nucleation and resulted in an adult heart with a greater number of small cardiac myocytes. These results suggest that TTCCs are involved in the regulation of myocyte size and the exit of myocytes from the cell cycle during the first week after birth.
PMCID: PMC3888788  PMID: 23743021
T-type Ca2+ channels; Cardiac myocytes; Cell cycle
9.  Bone morphogenetic protein 2 inhibits the proliferation and growth of human colorectal cancer cells 
Oncology Reports  2014;32(3):1013-1020.
Colorectal cancer (CRC) is one of the most deadly cancers worldwide. Significant progress has been made in understanding the molecular pathogenesis of CRC, which has led to successful early diagnosis, surgical intervention and combination chemotherapy. However, limited therapeutic options are available for metastatic and/or drug-resistant CRC. While the aberrantly activated Wnt/β-catenin pathway plays a critical initiating role in CRC development, disruption of the bone morphogenetic protein (BMP) pathway causes juvenile polyposis syndrome, suggesting that BMP signaling may play a role in CRC development. However, conflicting results have been reported concerning the possible roles of BMP signaling in sporadic colon cancer. Here, we investigated the effect of BMP2 on the proliferation, migration, invasiveness and tumor growth capability of human CRC cells. Using an adenovirus vector that overexpresses BMP2 and the piggyBac transposon-mediated stable BMP2 overexpression CRC line, we found that exogenous BMP2 effectively inhibited HCT116 cell proliferation and colony formation. BMP2 was shown to suppress colon cancer cell migration and invasiveness. Under a low serum culture condition, forced expression of BMP2 induced a significantly increased level of apoptosis in HCT116 cells. Using a xenograft tumor model, we found that forced expression of BMP2 in HCT116 cells suppressed tumor growth, accompanied by decreased cell proliferation activity. Taken together, our results strongly suggest that BMP2 plays an important inhibitory role in governing the proliferation and aggressive features of human CRC cells.
PMCID: PMC4121423  PMID: 24993644
colorectal cancer; BMP2; BMP signaling; tumorigenesis; proliferation; intestinal epithelial cells
10.  Nerve growth factor improves functional recovery by inhibiting endoplasmic reticulum stress-induced neuronal apoptosis in rats with spinal cord injury 
Endoplasmic reticulum (ER) stress-induced apoptosis plays a major role in various diseases, including spinal cord injury (SCI). Nerve growth factor (NGF) show neuroprotective effect and improve the recovery of SCI, but the relations of ER stress-induced apoptosis and the NGF therapeutic effect in SCI still unclear.
Young adult female Sprague-Dawley rats’s vertebral column was exposed and a laminectomy was done at T9 vertebrae and moderate contusion injuries were performed using a vascular clip. NGF stock solution was diluted with 0.9% NaCl and administered intravenously at a dose of 20 μg/kg/day after SCI and then once per day until they were executed. Subsequently, the rats were executed at 1d, 3 d, 7d and 14d. The locomotor activities of SCI model rats were tested by the 21-point Basso-Beattie-Bresnahan (BBB) locomotion scale, inclined plane test and footprint analysis. In addition, Western blot analysis was performed to identify the expression of ER-stress related proteins including CHOP, GRP78 and caspase-12 both in vivo and in vitro. The level of cell apoptosis was determined by TUNEL in vivo and Flow cytometry in vitro. Relative downstream signals Akt/GSK-3β and ERK1/2were also analyzed with or without inhibitors in vitro.
Our results demonstrated that ER stress-induced apoptosis was involved in the injury of SCI model rats. NGF administration improved the motor function recovery and increased the neurons survival in the spinal cord lesions of the model rats. NGF decreases neuron apoptosis which measured by TUNEL and inhibits the activation of caspase-3 cascade. The ER stress-induced apoptosis response proteins CHOP, GRP78 and caspase-12 are inhibited by NGF treatment. Meanwhile, NGF administration also increased expression of growth-associated protein 43 (GAP43). The administration of NGF activated downstream signals Akt/GSK-3β and ERK1/2 in ER stress cell model in vitro.
The neuroprotective role of NGF in the recovery of SCI is related to the inhibition of ER stress-induced cell death via the activation of downstream signals, also suggested a new trend of NGF translational drug development in the central neural system injuries which involved in the regulation of chronic ER stress.
PMCID: PMC4039547  PMID: 24884850
Nerve growth factor; Endoplasmic reticulum stress; Spinal cord injury; Apoptosis; Akt/GSK-3β; ERK1/2
11.  Adenovirus-Mediated Efficient Gene Transfer into Cultured Three-Dimensional Organoids 
PLoS ONE  2014;9(4):e93608.
Three-dimensional organoids have been recently established from various tissue-specific progenitors (such as intestinal stem cells), induced pluripotent stem cells, or embryonic stem cells. These cultured self-sustaining stem cell–based organoids may become valuable systems to study the roles of tissue-specific stem cells in tissue genesis and disease development. It is thus conceivable that effective genetic manipulations in such organoids may allow us to reconstruct disease processes and/or develop novel therapeutics. Recombinant adenoviruses are one of the most commonly used viral vectors for in vitro and in vivo gene deliveries. In this study, we investigate if adenoviruses can be used to effectively deliver transgenes into the cultured “mini-gut” organoids derived from intestinal stem cells. Using adenoviral vectors that express fluorescent proteins, we demonstrate that adenoviruses can effectively deliver transgenes into the cultured 3-D “mini-gut” organoids. The transgene expression can last at least 10 days in the cultured organoids. As a proof-of-principle experiment, we demonstrate that adenovirus-mediated noggin expression effectively support the survival and self-renewal of mini-gut organoids, while adenovirus-mediated expression of BMP4 inhibits the self-sustainability and proliferation of the organoids. Thus, our results strongly suggest that adenovirus vectors can be explored as effective gene delivery vehicles to introduce genetic manipulations in 3-D organoids.
PMCID: PMC3973564  PMID: 24695466
12.  Apelin inhibits the proliferation and migration of rat PASMCs via the activation of PI3K/Akt/mTOR signal and the inhibition of autophagy under hypoxia 
Apelin is highly expressed in the lungs, especially in the pulmonary vasculature, but the functional role of apelin under pathological conditions is still undefined. Hypoxic pulmonary hypertension is the most common cause of acute right heart failure, which may involve the remodeling of artery and regulation of autophagy. In this study, we determined whether treatment with apelin regulated the proliferation and migration of rat pulmonary arterial smooth muscle cells (SMCs) under hypoxia, and investigated the underlying mechanism and the relationship with autophagy. Our data showed that hypoxia activated autophagy significantly at 24 hrs. The addition of exogenous apelin decreased the level of autophagy and further inhibited pulmonary arterial SMC (PASMC) proliferation via activating downstream phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/the mammalian target of Rapamycin (mTOR) signal pathways. The inhibition of the apelin receptor (APJ) system by siRNA abolished the inhibitory effect of apelin in PASMCs under hypoxia. This study provides the evidence that exogenous apelin treatment contributes to inhibit the proliferation and migration of PASMCs by regulating the level of autophagy.
PMCID: PMC3955159  PMID: 24447518
apelin; hypoxia; autophagy; PI3K/Akt/mTOR; smooth muscle cells
13.  Identification of Cross-Species Shared Transcriptional Networks of Diabetic Nephropathy in Human and Mouse Glomeruli 
Diabetes  2012;62(1):299-308.
Murine models are valuable instruments in defining the pathogenesis of diabetic nephropathy (DN), but they only partially recapitulate disease manifestations of human DN, limiting their utility. To define the molecular similarities and differences between human and murine DN, we performed a cross-species comparison of glomerular transcriptional networks. Glomerular gene expression was profiled in patients with early type 2 DN and in three mouse models (streptozotocin DBA/2, C57BLKS db/db, and eNOS-deficient C57BLKS db/db mice). Species-specific transcriptional networks were generated and compared with a novel network-matching algorithm. Three shared human–mouse cross-species glomerular transcriptional networks containing 143 (Human-DBA STZ), 97 (Human-BKS db/db), and 162 (Human-BKS eNOS−/− db/db) gene nodes were generated. Shared nodes across all networks reflected established pathogenic mechanisms of diabetes complications, such as elements of Janus kinase (JAK)/signal transducer and activator of transcription (STAT) and vascular endothelial growth factor receptor (VEGFR) signaling pathways. In addition, novel pathways not previously associated with DN and cross-species gene nodes and pathways unique to each of the human–mouse networks were discovered. The human–mouse shared glomerular transcriptional networks will assist DN researchers in selecting mouse models most relevant to the human disease process of interest. Moreover, they will allow identification of new pathways shared between mice and humans.
PMCID: PMC3526018  PMID: 23139354
14.  Ca2+ influx through L-type Ca2+ channels and transient receptor potential channels activate pathological hypertrophy signaling 
Common cardiovascular diseases such as hypertension and myocardial infarction require that myocytes develop greater than normal force to maintain cardiac pump function. This requires increases in [Ca2+]. These diseases induce cardiac hypertrophy and increases in [Ca2+] are known to be an essential proximal signal for activation of hypertrophic genes. However, the source of “hypertrophic” [Ca2+] is not known and is the topic of this study. The role of Ca2+ influx through L-type Ca2+ channels (LTCC), T-type Ca2+ channels (TTCC) and transient receptor potential (TRP) channels on the activation of Calcineurin (Cn) – Nuclear Factor of Activated T cells (NFAT) signaling and myocyte hypertrophy was studied. Neonatal rat (NRVMs) and adult feline (AFVM) ventricular myocytes were infected with an adenovirus containing NFAT-GFP, to determine factors that could induce NFAT nuclear translocation. Four millimolar Ca2+ or pacing induced NFAT nuclear translocation. This effect was blocked by Cn inhibitors. In NRVMs Nifedipine (Nif, LTCC antagonist) blocked high Ca2+-induced NFAT nuclear translocation while SKF-96365 (TRP channel antagonist) and Nickel (Ni, TTCC antagonist) were less effective. The relative potency of these antagonists against Ca2+ induced NFAT nuclear translocation (Nif>SKF-96365>Ni) was similar to their effects on Ca2+ transients and the LTCC current. Infection of NRVM with viruses containing TRP channels also activated NFAT-GFP nuclear translocation and caused myocyte hypertrophy. TRP effects were reduced by SKF-96365, but were more effectively antagonized by Nif. These experiments suggest that Ca2+ influx through LTCCs is the primary source of Ca2+ to activate Cn-NFAT signaling in NRVMs and AFVMs. While TRP channels cause hypertrophy, they appear to do so through a mechanism involving Ca2+ entry via LTCCs.
PMCID: PMC3472041  PMID: 22921230
L-type calcium channel; Hypertrophy; Nuclear factor of activated T cells; Ventricular myocyte; Transient receptor potential channel
15.  Photoacoustic detection of functional responses in the motor cortex of awake behaving monkey during forelimb movement 
Journal of Biomedical Optics  2012;17(11):110503.
Photoacoustic (PA) imaging was applied to detect the neuronal activity in the motor cortex of an awake, behaving monkey during forelimb movement. An adult macaque monkey was trained to perform a reach-to-grasp task while PA images were acquired through a 30-mm diameter implanted cranial chamber. Increased PA signal amplitude results from an increase in regional blood volume and is interpreted as increased neuronal activity. Additionally, depth-resolved PA signals enabled the study of functional responses in deep cortical areas. The results demonstrate the feasibility of utilizing PA imaging for studies of functional activation of cerebral cortex in awake monkeys performing behavioral tasks.
PMCID: PMC3477655  PMID: 23089667
photoacoustic imaging; monkey; motor cortex; muscle; functional imaging
16.  Tongue force and tongue motility are differently affected by unilateral vs bilateral nigrostriatal dopamine depletion in rats 
Behavioural brain research  2012;234(2):343-348.
In addition to its cardinal symptoms of bradykinesia, muscle rigidity, resting tremor and postural disturbances, Parkinson’s disease (PD) also affects orolingual motor function. Orolingual motor deficits can contribute to dysphagia, which increases morbidity and mortality in this population. Previous preclinical studies describing orolingual motor deficits in animal models of PD have focused on unilateral nigrostriatal dopamine (DA) depletion. In this study we compared the effects of unilateral vs bilateral 6-hydroxydopamine (6-OHDA)-induced DA depletion in rats trained to lick water from an isometric force-sensing disc. Rats received either unilateral or bilateral 6-OHDA into the medial forebrain bundle and were tested for four weeks post-lesion. Dependent variables included task engagement (the number of licks per session), tongue force (mean and maximum), and tongue motility (the number of licks per second). While both lesion groups exhibited decreased tongue force output, tongue motility deficits were present in only the group that received unilateral nigrostriatal DA depletion. Task engagement was not significantly diminished by 6-OHDA. Analysis of striatal DA tissue content revealed that DA depletion was ~97% in the unilateral group and ~90% in the bilateral group. These results suggest that while nigrostriatal DA depletion affects tongue force output, deficits in tongue motility may instead result from a functional imbalance in neural pathways affecting this midline structure.
PMCID: PMC3422433  PMID: 22796604
Parkinson’s disease; orolingual; oromotor; isometric; operant; behavioral; nigrostriatal; dysarthria; dysphagia; force
17.  Endoplasmic Reticulum (ER) Stress Inducible Factor Cysteine-Rich with EGF-Like Domains 2 (Creld2) Is an Important Mediator of BMP9-Regulated Osteogenic Differentiation of Mesenchymal Stem Cells 
PLoS ONE  2013;8(9):e73086.
Mesenchymal stem cells (MSCs) are multipotent progenitors that can undergo osteogenic differentiation under proper stimuli. We demonstrated that BMP9 is one of the most osteogenic BMPs. However, the molecular mechanism underlying BMP9-initiated osteogenic signaling in MSCs remains unclear. Through gene expression profiling analysis we identified several candidate mediators of BMP9 osteogenic signaling. Here, we focus on one such signaling mediator and investigate the functional role of cysteine-rich with EGF-like domains 2 (Creld2) in BMP9-initiated osteogenic signaling. Creld2 was originally identified as an ER stress-inducible factor localized in the ER-Golgi apparatus. Our genomewide expression profiling analysis indicates that Creld2 is among the top up-regulated genes in BMP9-stimulated MSCs. We confirm that Creld2 is up-regulated by BMP9 in MSCs. ChIP analysis indicates that Smad1/5/8 directly binds to the Creld2 promoter in a BMP9-dependent fashion. Exogenous expression of Creld2 in MSCs potentiates BMP9-induced early and late osteogenic markers, and matrix mineralization. Conversely, silencing Creld2 expression inhibits BMP9-induced osteogenic differentiation. In vivo stem cell implantation assay reveals that exogenous Creld2 promotes BMP9-induced ectopic bone formation and matrix mineralization, whereas silencing Creld2 expression diminishes BMP9-induced bone formation and matrix mineralization. We further show that Creld2 is localized in ER and the ER stress inducers potentiate BMP9-induced osteogenic differentiation. Our results strongly suggest that Creld2 may be directly regulated by BMP9 and ER stress response may play an important role in regulating osteogenic differentiation.
PMCID: PMC3760886  PMID: 24019898
18.  Cross-talk between EGF and BMP9 signalling pathways regulates the osteogenic differentiation of mesenchymal stem cells 
Mesenchymal stem cells (MSCs) are multipotent progenitors, which give rise to several lineages, including bone, cartilage and fat. Epidermal growth factor (EGF) stimulates cell growth, proliferation and differentiation. EGF acts by binding with high affinity to epidermal growth factor receptor (EGFR) on the cell surface and stimulating the intrinsic protein tyrosine kinase activity of its receptor, which initiates a signal transduction cascade causing a variety of biochemical changes within the cell and regulating cell proliferation and differentiation. We have identified BMP9 as one of the most osteogenic BMPs in MSCs. In this study, we investigate if EGF signalling cross-talks with BMP9 and regulates BMP9-induced osteogenic differentiation. We find that EGF potentiates BMP9-induced early and late osteogenic markers of MSCs in vitro, which can be effectively blunted by EGFR inhibitors Gefitinib and Erlotinib or receptor tyrosine kinase inhibitors AG-1478 and AG-494 in a dose- and time-dependent manner. Furthermore, EGF significantly augments BMP9-induced bone formation in the cultured mouse foetal limb explants. In vivo stem cell implantation experiment reveals that exogenous expression of EGF in MSCs can effectively potentiate BMP9-induced ectopic bone formation, yielding larger and more mature bone masses. Interestingly, we find that, while EGF can induce BMP9 expression in MSCs, EGFR expression is directly up-regulated by BMP9 through Smad1/5/8 signalling pathway. Thus, the cross-talk between EGF and BMP9 signalling pathways in MSCs may underline their important roles in regulating osteogenic differentiation. Harnessing the synergy between BMP9 and EGF should be beneficial for enhancing osteogenesis in regenerative medicine.
PMCID: PMC4118175  PMID: 23844832
BMP9 signalling; osteoblastic differentiation; EGF signalling; mesenchymal stem cells; osteogenic differentiation
19.  Pancreaticoduodenectomy Combined with Vascular Resection and Reconstruction for Patients with Locally Advanced Pancreatic Cancer: A Multicenter, Retrospective Analysis 
PLoS ONE  2013;8(8):e70340.
The aim of this study was to present the therapeutic outcome of patients with locally advanced pancreatic cancer treated with pancreatoduodenectomy combined with vascular resection and reconstruction in addition to highlighting the mortality/morbidity and main prognostic factors associated with this treatment.
Materials and Methods
We retrospectively analyzed the clinical and pathological data of a total of 566 pancreatic cancer patients who were treated with PD from five teaching hospitals during the period of December 2006–December 2011. This study included 119 (21.0%) patients treated with PD combined with vascular resection and reconstruction. We performed a detailed statistical analysis of various factors, including postoperative complications, operative mortality, survival rate, operative time, pathological type, and lymph node metastasis.
The median survival time of the 119 cases that received PD combined with vascular resection was 13.3 months, and the 1-, 2-, and 3-year survival rates were 30.3%, 14.1%, and 8.1%, respectively. The postoperative complication incidence was 23.5%, and the mortality rate was 6.7%. For the combined vascular resection group, complications occurred in 28 cases (23.5%). For the group without vascular resection, complications occurred in 37 cases (8.2%). There was significant difference between the two groups (p = 0.001). The degree of tumor differentiation and the occurrence of complications after surgery were independent prognostic factors that determined the patients’ long-term survival.
Compared with PD without vascular resection, PD combined with vascular resection and reconstruction increased the incidence of postoperative complications. However, PD combined with vascular resection and reconstruction could achieve the complete removal of tumors without significantly increasing the mortality rate, and the median survival time was higher than that of patients who underwent palliative treatment. In addition, the two independent factors affecting the postoperative survival time were the degree of tumor differentiation and the presence or absence of postoperative complications.
PMCID: PMC3732270  PMID: 23936411
20.  Identification and Expression Profile Analysis of Odorant Binding Proteins in the Oriental Fruit Fly Bactrocera dorsalis 
Olfaction is crucial in many insects for critical behaviors, including those regulating survival and reproduction. Insect odorant-binding proteins (OBPs) function in the first step of the olfactory system and play an essential role in the perception of odorants, such as pheromones and host chemicals. The oriental fruit fly, Bactrocera dorsalis, is a destructive fruit-eating pest, due to its wide host range of up to 250 different types of fruits and vegetables, and this fly causes severe economic damage to the fruit and vegetable industry. However, OBP genes have not been largely identified in B. dorsalis. Based on our previously constructed B. dorsalis cDNA library, ten OBP genes were identified in B. dorsalis for the first time. A phylogenetic tree was generated to show the relationships among the 10 OBPs of B. dorsalis to OBP sequences of two other Dipteran species, including Drosophila melanogaster and the mosquito Anopheles gambiae. The expression profiles of the ten OBPs in different tissues (heads, thoraxes, abdomens, legs, wings, male antennae and female antenna) of the mated adults were analyzed by real-time PCR. The results showed that nine of them are highly expressed in the antenna of both sexes, except BdorOBP7. Four OBPs (BdorOBP1, BdorOBP4, BdorOBP8, and BdorOBP10) are also enriched in the abdomen, and BdorOBP7 is specifically expressed in leg, indicating that it may function in other biological processes. This work will provide insight into the roles of OBPs in chemoreception and help develop new pest-control strategies.
PMCID: PMC3742281  PMID: 23867609
odorant binding protein; olfaction; tissue expression pattern; Bactrocera dorsalis
21.  Ginseng saponin metabolite 20(S)-protopanaxadiol inhibits tumor growth by targeting multiple cancer signaling pathways 
Oncology Reports  2013;30(1):292-298.
Plant-derived active constituents and their semi-synthetic or synthetic analogs have served as major sources of anticancer drugs. 20(S)-protopanaxadiol (PPD) is a metabolite of ginseng saponin of both American ginseng (Panax quinquefolius L.) and Asian ginseng (Panax ginseng C.A. Meyer). We previously demonstrated that ginsenoside Rg3, a glucoside precursor of PPD, exhibits anti-proliferative effects on HCT116 cells and reduces tumor size in a xenograft model. Our subsequent study indicated that PPD has more potent antitumor activity than that of Rg3 in vitro although the mechanism underlying the anticancer activity of PPD remains to be defined. Here, we investigated the mechanism underlying the anticancer activity of PPD in human cancer cells in vitro and in vivo. PPD was shown to inhibit growth and induce cell cycle arrest in HCT116 cells. The in vivo studies indicate that PPD inhibits xenograft tumor growth in athymic nude mice bearing HCT116 cells. The xenograft tumor size was significantly reduced when the animals were treated with PPD (30 mg/kg body weight) for 3 weeks. When the expression of previously identified Rg3 targets, A kinase (PRKA) anchor protein 8 (AKAP8L) and phosphatidylinositol transfer protein α (PITPNA), was analyzed, PPD was shown to inhibit the expression of PITPNA while upregulating AKAP8L expression in HCT116 cells. Pathway-specific reporter assays indicated that PPD effectively suppressed the NF-κB, JNK and MAPK/ERK signaling pathways. Taken together, our results suggest that the anticancer activity of PPD in colon cancer cells may be mediated through targeting NF-κB, JNK and MAPK/ERK signaling pathways, although the detailed mechanisms underlying the anticancer mode of PPD action need to be fully elucidated.
PMCID: PMC3729206  PMID: 23633038
ginseng; ginseng metabolites; 20(S)-protopanaxadiol; colorectal cancer; signaling pathway; natural products
22.  Insights into the increasing virulence of the swine-origin pandemic H1N1/2009 influenza virus 
Scientific Reports  2013;3:1601.
Pandemic H1N1/2009 viruses have been stabilized in swine herds, and some strains display higher pathogenicity than the human-origin isolates. In this study, high-throughput RNA sequencing (RNA-seq) is applied to explore the systemic transcriptome responses of the mouse lungs infected by swine (Jia6/10) and human (LN/09) H1N1/2009 viruses. The transcriptome data show that Jia6/10 activates stronger virus-sensing signals, such as the toll-like receptor, RIG-I like receptor and NOD-like receptor signalings, as well as a stronger NF-κB and JAK-STAT singals, which play significant roles in inducing innate immunity. Most cytokines and interferon-stimulated genes show higher expression lever in Jia/06 infected groups. Meanwhile, virus Jia6/10 activates stronger production of reactive oxygen species, which might further promote higher mutation rate of the virus genome. Collectively, our data reveal that the swine-origin pandemic H1N1/2009 virus elicits a stronger innate immune reaction and pro-oxidation stimulation, which might relate closely to the increasing pathogenicity.
PMCID: PMC3615340  PMID: 23549303
23.  Hyperphosphorylation of the Cardiac Ryanodine Receptor at Serine 2808 Is Not Involved in Cardiac Dysfunction after Myocardial Infarction 
Circulation Research  2012;110(6):831-840.
Abnormal behavior of the cardiac ryanodine receptor (RyR2) has been linked to cardiac arrhythmias and heart failure (HF) after Myocardial infarction (MI). It has been proposed that protein kinase A (PKA) hyperphosphorylation of the RyR2 at a single residue, Ser-2808 is a critical mediator of RyR dysfunction, depressed cardiac performance and HF after MI.
We used a mouse model (RyRS2808A) in which PKA hyperphosphorylation of the RyR2 at Ser-2808 is prevented to determine whether loss of PKA phosphorylation at this site averts post MI cardiac pump dysfunction.
Methods and Results
MI was induced in WT and S2808A mice. Myocyte and cardiac function were compared in WT and S2808A animals before and after MI. The effects of the PKA activator Isoproterenol (Iso) on L-type Ca2+ current (ICaL), contractions and [Ca2+]I transients were also measured. Both WT and S2808A mice had depressed pump function after MI and were no differences between groups. MI size was also identical in both groups. L type Ca2+ current, contractions, Ca2+ transients and SR Ca2+ load were also not significantly different in WT versus S2808A myocytes either before or after MI. Iso effects on Ca2+ current, contraction, Ca2+ transients and SR Ca2+ load were identical in WT and S2808A myocytes before and after MI at both low and high concentrations.
These results strongly support the idea that PKA phosphorylation of RyR-S2808 is irrelevant to the development of cardiac dysfunction after MI, at least in the mice used in this study.
PMCID: PMC3322671  PMID: 22302785
Myocardial infarction; ryanodine receptor; heart failure; PKA hyperphosphorylation
24.  Draft Genome Sequence of Streptomyces bottropensis ATCC 25435, a Bottromycin-Producing Actinomycete 
Genome Announcements  2013;1(2):e00019-13.
A series of bottromycin antibiotics have been isolated and identified from Streptomyces bottropensis strain ATCC 25435. Here, a draft genome sequence of S. bottropensis ATCC 25435 is presented. The genome carries an intact biosynthetic gene cluster for bottromycin antibiotics, which provides insight into the combinatorial biosynthesis of bottromycin antibiotics.
PMCID: PMC3622962  PMID: 23516178
25.  BMP9 signaling in stem cell differentiation and osteogenesis 
Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily and play a critical role in skeletal development, bone formation and stem cell differentiation. Disruptions in BMP signaling result in a variety of skeletal and extraskeletal anomalies. BMP9 is a poorly characterized member of the BMP family and is among the most osteogenic BMPs, promoting osteoblastic differentiation of mesenchymal stem cells (MSCs) both in vitro and in vivo. Recent findings from various in vivo and molecular studies strongly suggest that the mechanisms governing BMP9-mediated osteoinduction differ from other osteogenic BMPs. Many signaling pathways with diverse functions have been found to play a role in BMP9-mediated osteogenesis. Several of these pathways are also critical in the differentiation of other cell lineages, including adipocytes and chondrocytes. While BMP9 is known to be a potent osteogenic factor, it also influences several other pathways including cancer development, angiogenesis and myogenesis. Although BMP9 has been demonstrated as one of the most osteogenic BMPs, relatively little is known about the specific mechanisms responsible for these effects. BMP9 has demonstrated efficacy in promoting spinal fusion and bony non-union repair in animal models, demonstrating great translational promise. This review aims to summarize our current knowledge of BMP9-mediated osteogenesis by presenting recently completed work which may help us to further elucidate these pathways.
PMCID: PMC3636726  PMID: 23671813
BMP; BMP9; bone regeneration; IGF; osteogenesis; TGF-β; Wnt; signal transduction; mesenchymal stem cells

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