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1.  Nanolayer encapsulation of insulin-chitosan complexes improves efficiency of oral insulin delivery 
Current oral insulin formulations reported in the literature are often associated with an unpredictable burst release of insulin in the intestine, which may increase the risk for problematic hypoglycemia. The aim of the study was to develop a solution based on a nanolayer encapsulation of insulin-chitosan complexes to afford sustained release after oral administration. Chitosan/heparin multilayer coatings were deposited onto insulin-chitosan microparticulate cores in the presence of poly(ethylene) glycol (PEG) in the precipitating and coating solutions. The addition of PEG improved insulin loading and minimized an undesirable loss of the protein resulting from redissolution. Nanolayer encapsulation and the formation of complexes enabled a superior loading capacity of insulin (>90%), as well as enhanced stability and 74% decreased solubility at acid pH in vitro, compared with nonencapsulated insulin. The capsulated insulin administered by oral gavage lowered fasting blood glucose levels by up to 50% in a sustained and dose-dependent manner and reduced postprandial glycemia in streptozotocin-induced diabetic mice without causing hypoglycemia. Nanolayer encapsulation reduced the possibility of rapid and erratic falls of blood glucose levels in animals. This technique represents a promising strategy to promote the intestinal absorption efficiency and release behavior of the hormone, potentially enabling an efficient and safe route for oral insulin delivery of insulin in diabetes management.
doi:10.2147/IJN.S59075
PMCID: PMC4014370  PMID: 24833901
oral insulin; diabetes mellitus; insulin-chitosan complexes; multilayer nanoencapsulation; polyethylene glycol; chitosan; heparin
2.  Tenascin-C Deficiency in Apo E−/− Mouse Increases Eotaxin Levels: Implications for Atherosclerosis 
Atherosclerosis  2013;227(2):267-274.
Aim
To investigate the potential role of inflammatory cytokines in apo E−/− mouse in response to deletion of Tenascin-C (TNC) gene.
Methods and results
We used antibody array and ELISA to compare the profile of circulating inflammatory cytokines in apo E−/− mice and apo E−/− TNC−/− double knockout mice. In addition, tissue culture studies were performed to investigate the activity of cells from each mouse genotype in vitro. Cytokine array analysis and subsequent ELISA showed that circulating eotaxin levels were selectively and markedly increased in response to TNC gene deletion in apo E−/− mice. In addition, considerable variation was noted in the circulating level of eotaxin among the control apo E−/− mouse group. Inbreeding of apo E−/− mice with high or low levels of plasma eotaxin showed that the level of eotaxin per se determines the extent of atherosclerosis in this mouse genotype. While endothelial cells from apo E−/− mice had low level of eotaxin expression, cells derived from apo E−/−TNC−/− mice expressed a high level of eotaxin. Transient transfection of eotaxin promoter-reporter constructs revealed that eotaxin expression is regulated at the transcriptional level by TNC. Histochemical analysis of aortic sections revealed the massive accumulation of mast cells in the adventitia of double KO mice lesions whereas no such accumulation was detected in the control group. Plasma from the apo E−/−TNC−/− mice markedly stimulated mast cell migration whereas plasma from the apo E−/− mice had no such effect.
Conclusion
These observations support the emerging hypothesis that TNC expression controls eotaxin level in apo E−/− mice and that this chemokine plays a key role in the development of atherosclerosis.
doi:10.1016/j.atherosclerosis.2013.01.039
PMCID: PMC3621962  PMID: 23433402
Tenascin-C; Eotaxin; Atherosclerosis; Mast cells; Chemokines; Matricellular protein; Extracellular matrix
3.  Genome-wide multi-omics profiling of colorectal cancer identifies immune determinants strongly associated with relapse 
Frontiers in Genetics  2013;4:236.
The use and benefit of adjuvant chemotherapy to treat stage II colorectal cancer (CRC) patients is not well understood since the majority of these patients are cured by surgery alone. Identification of biological markers of relapse is a critical challenge to effectively target treatments to the ~20% of patients destined to relapse. We have integrated molecular profiling results of several “omics” data types to determine the most reliable prognostic biomarkers for relapse in CRC using data from 40 stage I and II CRC patients. We identified 31 multi-omics features that highly correlate with relapse. The data types were integrated using multi-step analytical approach with consecutive elimination of redundant molecular features. For each data type a systems biology analysis was performed to identify pathways biological processes and disease categories most affected in relapse. The biomarkers detected in tumors urine and blood of patients indicated a strong association with immune processes including aberrant regulation of T-cell and B-cell activation that could lead to overall differences in lymphocyte recruitment for tumor infiltration and markers indicating likelihood of future relapse. The immune response was the biologically most coherent signature that emerged from our analyses among several other biological processes and corroborates other studies showing a strong immune response in patients less likely to relapse.
doi:10.3389/fgene.2013.00236
PMCID: PMC3834519  PMID: 24312117
colorectal cancer; relapse; variant analysis; integrative analysis; multi-omics; exome sequencing; systems biology; immune response
4.  High expression of astrocyte elevated gene-1 (AEG-1) is associated with progression of cervical intraepithelial neoplasia and unfavorable prognosis in cervical cancer 
Background
Astrocyte elevated gene-1(AEG-1) plays an important role in the development and progression of certain types of human cancers. However, the expression dynamics of AEG-1 in cervical cancer and its clinical/prognostic significance are unclear.
Method
In present study, the methods of tissue microarrays (TMA) and immunohistochemistry (IHC) were utilized to investigate AEG-1 expression in cervical intraepithelial neoplasia (CIN) and cervical cancer. Receiver operating characteristic (ROC) curve analysis, χ2 test, Kaplan-Meier plots, and multivariate Cox regression analysis were used to analyze the data.
Results
The expression level of AEG-1 was increased from CIN I to CIN III. High expression of AEG-1 could be observed in 61.1% (55/90) of cervical cancer. Moreover, high expression of AEG-1 correlated with tumor size and lymph node metastasis (all P <0.05). More importantly, high expression of AEG-1 was closely associated with cervical cancer patient shortened survival time as evidenced by univariate and multivariate analysis (P <0.05).
Conclusions
Our data suggest for the first time that high expression of AEG-1 is associated significantly with progression of cervical cancer. AEG-1 overexpression, as examined by IHC, has the potential to be used as an immunomarker to predict prognosis of cervical cancer patients.
doi:10.1186/1477-7819-11-297
PMCID: PMC3866971  PMID: 24256614
AEG-1; Cervical cancer; Tissue microarrays; Prognosis
5.  Identification of Molecular Pathway Aberrations in Uterine Serous Carcinoma by Genome-wide Analyses 
Background
Uterine cancer is the fourth most common malignancy in women, and uterine serous carcinoma is the most aggressive subtype. However, the molecular pathogenesis of uterine serous carcinoma is largely unknown. We analyzed the genomes of uterine serous carcinoma samples to better understand the molecular genetic characteristics of this cancer.
Methods
Whole-exome sequencing was performed on 10 uterine serous carcinomas and the matched normal blood or tissue samples. Somatically acquired sequence mutations were further verified by Sanger sequencing. The most frequent molecular genetic changes were further validated by Sanger sequencing in 66 additional uterine serous carcinomas and in nine serous endometrial intraepithelial carcinomas (the preinvasive precursor of uterine serous carcinoma) that were isolated by laser capture microdissection. In addition, gene copy number was characterized by single-nucleotide polymorphism (SNP) arrays in 23 uterine serous carcinomas, including 10 that were subjected to whole-exome sequencing.
Results
We found frequent somatic mutations in TP53 (81.6%), PIK3CA (23.7%), FBXW7 (19.7%), and PPP2R1A (18.4%) among the 76 uterine serous carcinomas examined. All nine serous carcinomas that had an associated serous endometrial intraepithelial carcinoma had concordant PIK3CA, PPP2R1A, and TP53 mutation status between uterine serous carcinoma and the concurrent serous endometrial intraepithelial carcinoma component. DNA copy number analysis revealed frequent genomic amplification of the CCNE1 locus (which encodes cyclin E, a known substrate of FBXW7) and deletion of the FBXW7 locus. Among 23 uterine serous carcinomas that were subjected to SNP array analysis, seven tumors with FBXW7 mutations (four tumors with point mutations, three tumors with hemizygous deletions) did not have CCNE1 amplification, and 13 (57%) tumors had either a molecular genetic alteration in FBXW7 or CCNE1 amplification. Nearly half of these uterine serous carcinomas (48%) harbored PIK3CA mutation and/or PIK3CA amplification.
Conclusion
Molecular genetic aberrations involving the p53, cyclin E–FBXW7, and PI3K pathways represent major mechanisms in the development of uterine serous carcinoma.
doi:10.1093/jnci/djs345
PMCID: PMC3692380  PMID: 22923510
6.  miR-3928 activates ATR pathway by targeting Dicer 
RNA Biology  2012;9(10):1247-1254.
Alterations in microRNA (miRNA) expression have been observed in cells subjected to exogenous stresses, implying that miRNAs play an important role in cellular stress response; however, the underlying mechanism is still largely unknown. In the present study, we found that miR-3928 was implicated in cellular response to ionizing radiation. After exposed to X-rays, miR-3928 expression increased in 1.5 h and then decreased, meanwhile Dicer, a key component in the miRNA processing machinery, increased gradually. An oscillation was observed in the expression of both mature miR-3928 and Dicer mRNA from 2 h to 3.5 h in irradiated cells. Then, we verified that miR-3928 directly bound to the 3'-untranslated region of Dicer mRNA. Consequently, Dicer expression was suppressed and the maturation of other miRNAs including miR-185, miR-300, and miR-663, was inhibited. Overexpression of miR-3928 induced DNA damage, activated ATR, and phosphorylated Chk1 accompanied by G1 arrest. Taken together, these findings replenished ATR/Chk1 pathway by revealing a novel miRNA regulatory network in response to exogenous stress, in which miR-3928 plays an important role in regulating the expression of Dicer.
doi:10.4161/rna.21821
PMCID: PMC3583855  PMID: 22922797
DNA damage; Dicer; G1 arrest; ionizing radiation; microRNA
7.  Deletion of Tenascin-C gene Exacerbates Atherosclerosis and Induces Intraplaque Hemorrhage in Apo E Deficient Mice 
Aims
Tenascin-C (TNC), a matricellular protein, is upregulated in atherosclerotic plaques. We investigated whether the deletion of TNC gene affects the development of atherosclerosis in a murine model.
Methods
TNC−/−/apo E−/− mice were generated and used for atherosclerosis studies. We compared these results to those observed in control groups of apo E−/− mice.
Results
The en face analysis of aortic area showed that the mean aortic lesion area of the double KO mice was significantly higher than control mice at different times after feeding of atherogenic diet; the accumulation of lesional macrophages and lipids were significantly higher, respectively. Analysis of cell adhesion molecules revealed that VCAM-1, but not ICAM-1, was upregulated 1 week after feeding of atherogenic diet in the double KO mouse as compared to apo E−/− mouse. Cell culture studies revealed that the expression of VCAM-1 in endothelial cells isolated from the double KO mouse is more sensitive to the TNFα stimulation than the cells isolated from apo E−/− mice. Cell adhesion studies showed that the adherence of RAW monocytic cells to the endothelial cells was significantly enhanced in the cultured endothelial cells from the TNC gene-deleted cells. Following the prolonged feeding of an atherogenic diet (28–30 weeks), the aortic and carotid atherosclerotic lesions frequently demonstrated large grossly visible areas of intraplaque hemorrhage in the double KO mice compared to control.
Conclusions
These data unveil a protective role for TNC in atherosclerosis and suggest that TNC signaling may have the potential to reduce atherosclerosis, in part by modulating VCAM-1 expression.
doi:10.1016/j.carpath.2011.12.005
PMCID: PMC3345312  PMID: 22300502
tenascin; atherosclerosis; plaque hemorrhage; extracellular matrix; VCAM-1
8.  The potential value of the neutral comet assay and γH2AX foci assay in assessing the radiosensitivity of carbon beam in human tumor cell lines 
Radiology and Oncology  2013;47(3):247-257.
Background
Carbon ions (12C6+) are high linear energy transfer (LET) radiation characterized by higher relative biological effectiveness than low LET radiation. The assessment of tumour radiosensitivity would be particularly useful in optimizing the radiation dose during radiotherapy. The aim of the current study was to evaluate the potential value of the neutral comet assay and γH2AX foci assay in assessing 12C6+ radiosensitivity of tumour cells.
Materials and methods
The doses of 12C6+ and X-rays used in the present study were 2 and 4 Gy. The survival fraction, DNA double-strand breaks (DSB) and repair kinetics of DSB were assayed with clonogenic survival, neutral comet assay and γH2AX foci assay in human cervical carcinoma HeLa cells, hepatoma HepG2 cells, and mucoepidermoid carcinoma MEC-1 cells at the time points of 0.5, 4, 16 and 24 h after 12C6+ and X-rays irradiation.
Results
The survival fraction for 12C6+ irradiation was much more inhibited than for X-rays (p < 0.05) in all three tumour cell lines tested. Substantial amounts of residual damage, assessed by the neutral comet assay, were present after irradiation (p < 0.05). The highest residual damage was observed at 0.5 or 4 h, both for 12C6+ and X-ray irradiation. However, the residual damage in HeLa and MEC-1 cells was higher for 12C6+ than X-rays (p < 0.05). The strongest induction of γH2AX foci was observed after 30 min, for all three tumour cell lines (p < 0.01). The franction of γH2AX foci persisted for at least 24 h after 12C6+ irradiation; in HeLa cells and MEC-1 was higher than after X-ray irradiation (p < 0.05). The correlation coefficients between the clonogenic survival, neutral comet assay and γH2AX foci assay were not statistically significant, except for some tumour cells at individual irradiation doses and types.
Conclusions
Our study demonstrated that the neutral comet assay and γ-H2AX foci assay could be used to assess the radiosensitivity of 12C6+ in human tumour cells.
doi:10.2478/raon-2013-0045
PMCID: PMC3794881  PMID: 24133390
human tumour cells; carbon ions; X-rays; radiation sensitivity; DNA double strand breaks; γH2AX
9.  Patch-Clamp Recordings from Lateral Line Neuromast Hair Cells of the Living Zebrafish 
Zebrafish are popular models for biological discovery. For investigators of the auditory and vestibular periphery, manipulations of hair cell and synaptic mechanisms have relied on inferences from extracellular recordings of physiological activity. We now provide data showing that hair cells and supporting cells of the lateral line can be directly patch-clamped, providing the first recordings of ionic channel activity, synaptic vesicle release, and gap junctional coupling in the neuromasts of living fish. Such capabilities will allow more detailed understanding of mechano-sensation of the zebrafish.
doi:10.1523/JNEUROSCI.4265-12.2013
PMCID: PMC3684625  PMID: 23407967
10.  Targeting Rac1 Signaling Inhibits Streptococcal M1 Protein-Induced CXC Chemokine Formation, Neutrophil Infiltration and Lung Injury 
PLoS ONE  2013;8(8):e71080.
Infections with Streptococcus pyogenes exhibit a wide spectrum of infections ranging from mild pharyngitis to severe Streptococcal toxic shock syndrome (STSS). The M1 serotype of Streptococcus pyogenes is most commonly associated with STSS. In the present study, we hypothesized that Rac1 signaling might regulate M1 protein-induced lung injury. We studied the effect of a Rac1 inhibitor (NSC23766) on M1 protein-provoked pulmonary injury. Male C57BL/6 mice received NSC23766 prior to M1 protein challenge. Bronchoalveolar fluid and lung tissue were harvested for quantification of neutrophil recruitment, edema and CXC chemokine formation. Neutrophil expression of Mac-1 was quantified by use of flow cytometry. Quantitative RT-PCR was used to determine gene expression of CXC chemokines in alveolar macrophages. Treatment with NSC23766 decreased M1 protein-induced neutrophil infiltration, edema formation and tissue injury in the lung. M1 protein challenge markedly enhanced Mac-1 expression on neutrophils and CXC chemokine levels in the lung. Inhibition of Rac1 activity had no effect on M1 protein-induced expression of Mac-1 on neutrophils. However, Rac1 inhibition markedly decreased M1 protein-evoked formation of CXC chemokines in the lung. Moreover, NSC23766 completely inhibited M1 protein-provoked gene expression of CXC chemokines in alveolar macrophages. We conclude that these novel results suggest that Rac1 signaling is a significant regulator of neutrophil infiltration and CXC chemokine production in the lung. Thus, targeting Rac1 activity might be a potent strategy to attenuate streptococcal M1 protein-triggered acute lung damage.
doi:10.1371/journal.pone.0071080
PMCID: PMC3741375  PMID: 23951087
11.  Autosomal Recessive Transmission of MYBPC3 Mutation Results in Malignant Phenotype of Hypertrophic Cardiomyopathy 
PLoS ONE  2013;8(6):e67087.
Background
Hypertrophic cardiomyopathy (HCM) due to mutations in genes encoding sarcomere proteins is most commonly inherited as an autosomal dominant trait. Since nearly 50% of HCM cases occur in the absence of a family history, a recessive inheritance pattern may be involved.
Methods
A pedigree was identified with suspected autosomal recessive transmission of HCM. Twenty-six HCM-related genes were comprehensively screened for mutations in the proband with targeted second generation sequencing, and the identified mutation was confirmed with bi-directional Sanger sequencing in all family members and 376 healthy controls.
Results
A novel missense mutation (c.1469G>T, p.Gly490Val) in exon 17 of MYBPC3 was identified. Two siblings with HCM were homozygous for this mutation, whereas other family members were either heterozygous or wild type. Clinical evaluation showed that both homozygotes manifested a typical HCM presentation, but none of others, including 5 adult heterozygous mutation carriers up to 71 years of age, had any clinical evidence of HCM.
Conclusions
Our data identified a MYBPC3 mutation in HCM, which appeared autosomal recessively inherited in this family. The absence of a family history of clinical HCM may be due to not only a de novo mutation, but also recessive mutations that failed to produce a clinical phenotype in heterozygous family members. Therefore, consideration of recessive mutations leading to HCM is essential for risk stratification and genetic counseling.
doi:10.1371/journal.pone.0067087
PMCID: PMC3695947  PMID: 23840593
12.  Atorvastatin Enhance Efficacy of Mesenchymal Stem Cells Treatment for Swine Myocardial Infarction via Activation of Nitric Oxide Synthase 
PLoS ONE  2013;8(5):e65702.
Background
In a swine model of acute myocardial infarction (AMI), Statins can enhance the therapeutic efficacy of mesenchymal stem cell (MSCs) transplantation. However, the mechanisms remain unclear. This study aims at assessing whether atorvastatin (Ator) facilitates the effects of MSCs through activation of nitric oxide synthase (NOS), especially endothelial nitric oxide synthase (eNOS), which is known to protect against ischemic injury.
Methods and Results
42 miniswines were randomized into six groups (n = 7/group): Sham operation; AMI control; Ator only; MSC only, Ator+MSCs and Ator+MSCs+NG-nitrol-L-arginine (L-NNA), an inhibitor of NOS. In an open-heart surgery, swine coronary artery ligation and reperfusion model were established, and autologous bone-marrow MSCs were injected intramyocardium. Four weeks after transplantation, compared with the control group, Ator+MSCs animals exhibited decreased defect areas of both “perfusion” defined by Single-Photon Emission Computed Tomography (−6.2±1.8% vs. 2.0±5.1%, P = 0.0001) and “metabolism” defined by Positron Emission Tomography (−3.00±1.41% vs. 4.20±4.09%, P = 0.0004); Ejection fraction by Magnetic Resonance Imaging increased substantially (14.22±12.8% vs. 1.64±2.64%, P = 0.019). In addition, indices of inflammation, fibrosis, and apoptosis were reduced and survivals of MSCs or MSC-derived cells were increased in Ator+MSCs animals. In Ator or MSCs alone group, perfusion, metabolism, inflammation, fibrosis or apoptosis were reduced but there were no benefits in terms of heart function and cell survival. Furthermore, the above benefits of Ator+MSCs treatment could be partially blocked by L-NNA.
Conclusions
Atorvastatin facilitates survival of implanted MSCs, improves function and morphology of infarcted hearts, mediated by activation of eNOS and alleviated by NOS inhibitor. The data reveal the cellular and molecular mechanism for anti-AMI therapy with a combination of statin and stem cells.
doi:10.1371/journal.pone.0065702
PMCID: PMC3669282  PMID: 23741509
13.  Platform for Personalized Oncology: Integrative analyses reveal novel molecular signatures associated with colorectal cancer relapse 
Approximately 80% of Stage II colon cancer patients are cured by appropriate surgery. However, 20% relapse, and virtually all of these people will die due to metastatic disease. Adjuvant chemotherapy has little or no impact on relapse or survival in Stage II colon cancer, and can only add toxicity without benefit for 80% of the target population that has been cured by surgery. Despite much effort, it is difficult to identify clinical or molecular determinants of outcome in Stage II colon cancer, defeating attempts to target treatments to the 20% of individuals who are destined to relapse. We hypothesized that a multidimensional molecular analysis will identify a combination of factors that serve as prognostic biomarkers in Stage II adenocarcinoma of the colon. The Georgetown informatics team generated and analyzed multi-omics profiling datasets in stage II CRC patients with or without relapse to identify molecular signatures in CRC that may serve both as prognostic markers of recurrence, and also allow for identification of the subgroup of patients who might benefit from adjuvant chemotherapy. The datasets were loaded to GDOC® (Georgetown Database of Cancer) for further mining and analysis. The G-DOC web portal (http://gdoc.georgetown.edu) includes a broad collection of bioinformatics and systems biology tools for analysis and visualization of four major “omics” types: DNA, mRNA, microRNA, and metabolites. Through technology re-use, the G-DOC infrastructure will accelerate progress for a variety of ongoing programs in need of integrative multi-omics analysis, and advance our opportunities to practice effective personalized oncology in the near future.
PMCID: PMC3814476  PMID: 24303318
14.  TNNI3K, a Cardiac-Specific Kinase, Promotes Physiological Cardiac Hypertrophy in Transgenic Mice 
PLoS ONE  2013;8(3):e58570.
Purpose
Protein kinase plays an essential role in controlling cardiac growth and hypertrophic remodeling. The cardiac troponin I-interacting kinase (TNNI3K), a novel cardiac specific kinase, is associated with cardiomyocyte hypertrophy. However, the precise function of TNNI3K in regulating cardiac remodeling has remained controversial.
Methods and Results
In a rat model of cardiac hypertrophy generated by transverse aortic constriction, myocardial TNNI3K expression was significantly increased by 1.62 folds (P<0.05) after constriction for 15 days. To investigate the role of TNNI3K in cardiac hypertrophy, we generated transgenic mouse lines with overexpression of human TNNI3K specifically in the heart. At the age of 3 months, the high-copy-number TNNI3K transgenic mice demonstrated a phenotype of concentric hypertrophy with increased heart weight normalized to body weight (1.31 fold, P<0.01). Echocardiography and non-invasive hemodynamic assessments showed enhanced cardiac function. No necrosis or myocyte disarray was observed in the heart of TNNI3K transgenic mice. This concentric hypertrophy maintained up to 12 months of age without cardiac dysfunction. The phospho amino acid analysis revealed that TNNI3K is a protein-tyrosine kinase. The yeast two-hybrid screen and co-immunoprecipitation assay identified cTnI as a target for TNNI3K. Moreover, TNNI3K overexpression induced cTnI phosphorylation at Ser22/Ser23 in vivo and in vitro, suggesting that TNNI3K is a novel upstream regulator for cTnI phosphorylation.
Conclusion
TNNI3K promotes a concentric hypertrophy with enhancement of cardiac function via regulating the phosphorylation of cTnI. TNNI3K could be a potential therapeutic target for preventing from heart failure.
doi:10.1371/journal.pone.0058570
PMCID: PMC3589374  PMID: 23472207
15.  Pleiotrophin Induces Transdifferentiation of Monocytes Into Functional Endothelial Cells 
Objective
Pleiotrophin (PTN) is a cytokine that is expressed by monocytes/macrophages in ischemic tissues and that promotes neovascularization, presumably by stimulating proliferation of local endothelial cells. However, the effect of PTN on monocytes/macrophages remains unknown. We investigated the role of PTN in regulating the phenotype of monocytes/macrophages.
Methods and Results
RT-PCR, real-time PCR, and fluorescence-activated cell sorter analysis revealed that the expression of PTN by monocytic cells led to a downregulation of CD68, c-fms, and CD14 monocytic cell markers and an upregulation of FLK-1, Tie-2, vascular endothelial-cadherin, platelet endothelial cell adhesion molecule-1, endothelial NO synthase, von Willebrand factor, CD34, GATA-2, and GATA-3 endothelial cell markers. Fibrin gel assays showed that the treatment of mouse and human monocytic cells with PTN led to the formation of tube-like structures. In vivo studies showed that PTN-expressing monocytic cells incorporated into the blood vessels of the quail chorioallantoic membrane. The intracardial injection of PTN-expressing monocytic cells into chicken embryos showed that cells integrated only into the developing vasculature. Finally, the injection of PTN-expressing monocytes into a murine ischemic hindlimb model significantly improved perfusion of the ischemic tissue.
Conclusions
PTN expression by monocytes/macrophages led to a downregulation of their monocytic cell markers and an upregulation of endothelial cell characteristics, thus inducing the transdifferentiation of monocytes into functional endothelial cells.
doi:10.1161/01.ATV.0000222017.05085.8e
PMCID: PMC3579570  PMID: 16614316
transdifferentiation; pleiotrophin; macrophage; endothelial cell
16.  Mitochondrial stress engages E2F1 apoptotic signaling to cause deafness 
Cell  2012;148(4):716-726.
SUMMARY
Mitochondrial dysfunction causes poorly understood tissue-specific pathology stemming from primary defects in respiration, coupled with altered reactive oxygen species (ROS), metabolic signaling and apoptosis. The A1555G mtDNA mutation that causes maternally inherited deafness disrupts mitochondrial ribosome function, in part, via increased methylation of the mitochondrial 12S rRNA by the methyltransferase mtTFB1. In patient-derived A1555G cells, we show that 12S rRNA hyper-methylation causes ROS-dependent activation of AMP kinase and the pro-apoptotic nuclear transcription factor E2F1. This retrograde mitochondrial-stress relay is operative in vivo as transgenic-mtTFB1 mice exhibit enhanced 12S rRNA methylation in multiple tissues, increased E2F1 and apoptosis in the stria vascularis and spiral ganglion neurons of the inner ear, and progressive E2F1-dependent hearing loss. This transgenic-mtTFB1 mouse mitochondrial disease model provides a robust platform for deciphering the complex tissue-specificity of human mitochondrial-based disorders, as well as the precise pathogenic mechanism of maternally inherited deafness and its exacerbation by environmental factors.
doi:10.1016/j.cell.2011.12.027
PMCID: PMC3285425  PMID: 22341444
18.  Chemotherapy targeting by DNA capture in viral protein particles 
Nanomedicine (London, England)  2012;7(3):335-352.
Aim
This study tests the hypothesis that DNA intercalation and electrophilic interactions can be exploited to noncovalently assemble doxorubicin in a viral protein nanoparticle designed to target and penetrate tumor cells through ligand-directed delivery. We further test whether this new paradigm of doxorubicin targeting shows therapeutic efficacy and safety in vitro and in vivo.
Materials & methods
We tested serum stability, tumor targeting and therapeutic efficacy in vitro and in vivo using biochemical, microscopy and cytotoxicity assays.
Results
Self-assembly formed approximately 10-nm diameter serum-stable nanoparticles that can target and ablate HER2+ tumors at >10× lower dose compared with untargeted doxorubicin, while sparing the heart after intravenous delivery. The targeted nanoparticle tested here allows doxorubicin potency to remain unaltered during assembly, transport and release into target cells, while avoiding peripheral tissue damage and enabling lower, and thus safer, drug dose for tumor killing.
Conclusion
This nanoparticle may be an improved alternative to chemical conjugates and signal-blocking antibodies for tumor-targeted treatment.
doi:10.2217/nnm.11.104
PMCID: PMC3429352  PMID: 22385197
doxorubicin; HER; herdox; nanoparticle; noncovalent; penton base; self-assembly; tumor targeting; viral capsid
19.  Successful Development of Small Diameter Tissue-Engineering Vascular Vessels by Our Novel Integrally Designed Pulsatile Perfusion-Based Bioreactor 
PLoS ONE  2012;7(8):e42569.
Small-diameter (<4 mm) vascular constructs are urgently needed for patients requiring replacement of their peripheral vessels. However, successful development of constructs remains a significant challenge. In this study, we successfully developed small-diameter vascular constructs with high patency using our integrally designed computer-controlled bioreactor system. This computer-controlled bioreactor system can confer physiological mechanical stimuli and fluid flow similar to physiological stimuli to the cultured grafts. The medium circulating system optimizes the culture conditions by maintaining fixed concentration of O2 and CO2 in the medium flow and constant delivery of nutrients and waste metabolites, as well as eliminates the complicated replacement of culture medium in traditional vascular tissue engineering. Biochemical and mechanical assay of newly developed grafts confirm the feasibility of the bioreactor system for small-diameter vascular engineering. Furthermore, the computer-controlled bioreactor is superior for cultured cell proliferation compared with the traditional non-computer-controlled bioreactor. Specifically, our novel bioreactor system may be a potential alternative for tissue engineering of large-scale small-diameter vascular vessels for clinical use.
doi:10.1371/journal.pone.0042569
PMCID: PMC3411804  PMID: 22880036
20.  Metagenomic Insights into the Fibrolytic Microbiome in Yak Rumen 
PLoS ONE  2012;7(7):e40430.
The rumen hosts one of the most efficient microbial systems for degrading plant cell walls, yet the predominant cellulolytic proteins and fibrolytic mechanism(s) remain elusive. Here we investigated the cellulolytic microbiome of the yak rumen by using a combination of metagenome-based and bacterial artificial chromosome (BAC)-based functional screening approaches. Totally 223 fibrolytic BAC clones were pyrosequenced and 10,070 ORFs were identified. Among them 150 were annotated as the glycoside hydrolase (GH) genes for fibrolytic proteins, and the majority (69%) of them were clustered or linked with genes encoding related functions. Among the 35 fibrolytic contigs of >10 Kb in length, 25 were derived from Bacteroidetes and four from Firmicutes. Coverage analysis indicated that the fibrolytic genes on most Bacteroidetes-contigs were abundantly represented in the metagenomic sequences, and they were frequently linked with genes encoding SusC/SusD-type outer-membrane proteins. GH5, GH9, and GH10 cellulase/hemicellulase genes were predominant, but no GH48 exocellulase gene was found. Most (85%) of the cellulase and hemicellulase proteins possessed a signal peptide; only a few carried carbohydrate-binding modules, and no cellulosomal domains were detected. These findings suggest that the SucC/SucD-involving mechanism, instead of one based on cellulosomes or the free-enzyme system, serves a major role in lignocellulose degradation in yak rumen. Genes encoding an endoglucanase of a novel GH5 subfamily occurred frequently in the metagenome, and the recombinant proteins encoded by the genes displayed moderate Avicelase in addition to endoglucanase activities, suggesting their important contribution to lignocellulose degradation in the exocellulase-scarce rumen.
doi:10.1371/journal.pone.0040430
PMCID: PMC3396655  PMID: 22808161
21.  4-(4-Chloro­phen­yl)-2,6-bis­(1H-indol-3-yl)-1,4-dihydro­pyridine-3,5-dicarbo­nitrile ethanol monosolvate 
In the title compound, C29H18ClN5·C2H6O, the dihydro­pyridine ring adopts a strongly flattened envelope conformation, with a maximum deviation of 0.139 (2) Å from its best plane for the Csp 3 atom. The dihedral angles between the dihydro­pyridine ring plane and the two indole rings in positions 2 and 6 are 34.28 (5) and 40.50 (6)°, respectively. In turn, the benzene ring and the dihydro­pyridine ring are oriented at a dihedral angle of 74.69 (6)°. An intra­molecular C—H⋯Cl hydrogen bond occurs. In the crystal, mol­ecules are linked by N—H⋯N, N—H⋯O and O—H⋯N hydrogen bonds into layers parallel to (001). There are short C—H⋯Cl contacts between mol­ecules in neighboring layers.
doi:10.1107/S1600536812013906
PMCID: PMC3344446  PMID: 22590208
22.  Bioengineered vascular graft grown in the mouse peritoneal cavity 
Background
We tested the hypothesis that the mouse peritoneum can function like a bioreactor to generate directed bio-engineered tissues such as those used for bypass grafting. Additionally, we reasoned that the mouse animal model would allow us to elucidate the underlying cellular and molecular mechanisms that are responsible for the generation of tissue in peritoneal cavity.
Methods
Plastic tubes (2 tubes/mouse) were implanted into the peritoneal cavity of 3 strains of mice (C57BL/6, BALB/c, and MRL). The tubes were harvested, tissue capsule surrounding the tubes was removed, and analyzed by immunostaining (5 capsules/5 mice/strain) and microarray (3 capsules/3 mice/strain). In addition, the tissue capsules that were harvested from MRL mice (n=21) were grafted into abdominal aorta of the same mice as autografts. The patency of all grafts was monitored by micro-ultrasound and their functionality was assessed by Laser Doppler Imaging of blood flow in femoral arteries. Venous (n=13) and arterial isografts (n=11) were used as positive controls. In a negative control group (5 mice/strain), the abdominal aorta was occluded by double ligation with 9-0 silk.
Results
The implanted plastic tubes required at least 8 weeks of incubation in the peritoneum of the 3 strains of mice in order to generate useful grafts. No vascular cells. were found in the tissue capsules. Microarray analysis of tissue capsules revealed that the capsular cells express a gene expression program that is vastly shared among the 3 strains of mice and the cells exhibit high degree of plasticity. The micro-ultrasound analysis of the grafts showed that 62% of autografts remained patent compared to 77% of venous isografts and 91% of arterial isografts. The Laser Doppler Imaging analysis showed that blood flow dropped by 40% and 35% in the autografts and vein isografts, respectively, one day after surgery. The flow, however, rebounded to the level of arterial isografts one month post surgery and remained unchanged among all grafts for the next 4 months. Immunostaining of the autografts showed a thick vessel wall with endothelial cells that lined the lumen and smooth muscle cells that constituted the graft wall.
Conclusion
The mouse peritoneal cavity of mice has the ability to function like a bioreactor to generate bio-engineered tissues. The tissue capsules harvested from peritoneal cavity of a mouse are composed of nonvascular cells that display phenotype of progenitor cells. After grafting, however, the capsule auto-grafts become arterialized and remained patent for at least 4 months after surgery, similar to venous or arterial iso-grafts.
doi:10.1016/j.jvs.2010.05.015
PMCID: PMC2949474  PMID: 20692791
23.  Recent advances in nanotechnology based drug delivery to the brain 
Cytotechnology  2010;62(5):377-380.
Drug delivery into the brain was difficult due to the existence of blood brain barrier, which only permits some molecules to pass through freely. In past decades, nanotechnology has enabled many technical advances including drug delivery into the brain with high efficiency and accuracy. In the present paper, we summarize recent important advances in employing nanotechnology for drug delivery to the brain as well as controlled drug release.
doi:10.1007/s10616-010-9295-8
PMCID: PMC2993862  PMID: 20700653
Nanotechnology; Nanoparticle; Drug delivery; Blood–brain barrier
25.  G-DOC: A Systems Medicine Platform for Personalized Oncology1 
Neoplasia (New York, N.Y.)  2011;13(9):771-783.
Currently, cancer therapy remains limited by a “one-size-fits-all” approach, whereby treatment decisions are based mainly on the clinical stage of disease, yet fail to reference the individual's underlying biology and its role driving malignancy. Identifying better personalized therapies for cancer treatment is hindered by the lack of high-quality “omics” data of sufficient size to produce meaningful results and the ability to integrate biomedical data from disparate technologies. Resolving these issues will help translation of therapies from research to clinic by helping clinicians develop patient-specific treatments based on the unique signatures of patient's tumor. Here we describe the Georgetown Database of Cancer (G-DOC), a Web platform that enables basic and clinical research by integrating patient characteristics and clinical outcome data with a variety of high-throughput research data in a unified environment. While several rich data repositories for high-dimensional research data exist in the public domain, most focus on a single-data type and do not support integration across multiple technologies. Currently, G-DOC contains data from more than 2500 breast cancer patients and 800 gastrointestinal cancer patients, G-DOC includes a broad collection of bioinformatics and systems biology tools for analysis and visualization of four major “omics” types: DNA, mRNA, microRNA, and metabolites. We believe that G-DOC will help facilitate systems medicine by providing identification of trends and patterns in integrated data sets and hence facilitate the use of better targeted therapies for cancer. A set of representative usage scenarios is provided to highlight the technical capabilities of this resource.
PMCID: PMC3182270  PMID: 21969811

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