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1.  A high density of tertiary lymphoid structure B cells in lung tumors is associated with increased CD4+ T cell receptor repertoire clonality 
Oncoimmunology  2015;4(12):e1051922.
T and B cell receptor (TCR and BCR, respectively) Vβ or immunoglobulin heavy chain complementarity-determining region 3 sequencing allows monitoring of repertoire changes through recognition, clonal expansion, affinity maturation, and T or B cell activation in response to antigen. TCR and BCR repertoire analysis can advance understanding of antitumor immune responses in the tumor microenvironment. TCR and BCR repertoires of sorted CD4+, CD8+ or CD19+ cells in tumor, non-tumoral distant tissue (NT), and peripheral compartments (blood/draining lymph node [P]) from 47 non-small cell lung cancer (NSCLC) patients (agemedian = 68 y) were sequenced. The clonotype spectra were assessed among different tissues and correlated with clinical and immunological parameters. In all tissues, CD4+ and CD8+ TCR repertoires had greater clonality relative to CD19+ BCR. CD4+ T cells exhibited greater clonality in NT compared to tumor (p = 0.002) and P (p < 0.001), concentrated among older patients (age > 68). Younger patients exhibited greater CD4+ T cell diversity in P compared to older patients (p = 0.05), and greater CD4+ T cell clonality in tumor relative to P (p < 0.001), with fewer shared clonotypes between tumor and P than older patients (p = 0.04). More interestingly, greater CD4+ and CD8+ T cell clonality in tumor and P, respectively (both p = 0.05), correlated with high density of tumor-associated tertiary lymphoid structure (TLS) B cells, a biomarker of higher overall survival in NSCLC. Results indicate distinct adaptive immune responses in NSCLC, where peripheral T cell diversity is modulated by age, and tumor T cell clonal expansion is favored by the presence of TLSs in the tumor microenvironment.
doi:10.1080/2162402X.2015.1051922
PMCID: PMC4635865  PMID: 26587322
deep sequencing; non-small cell lung cancer; TCR/BCR repertoire; tertiary lymphoid structure
2.  Human Umbilical Cord Mesenchymal Stem Cell Exosomes Enhance Angiogenesis Through the Wnt4/β-Catenin Pathway 
Human umbilical cord mesenchymal stem cells (hucMSCs) and their exosomes have been considered as potential therapeutic tools for tissue regeneration; however, the underlying mechanisms are not well understood. The results of this study suggest that hucMSC-exosome-mediated Wnt4 induces β-catenin activation in endothelial cells and exerts proangiogenic effects, which could be an important mechanism for cutaneous wound healing.
Human umbilical cord mesenchymal stem cells (hucMSCs) and their exosomes have been considered as potential therapeutic tools for tissue regeneration; however, the underlying mechanisms are still not well understood. In this study, we isolated and characterized the exosomes from hucMSCs (hucMSC-Ex) and demonstrated that hucMSC-Ex promoted the proliferation, migration, and tube formation of endothelial cells in a dose-dependent manner. Furthermore, we demonstrated that hucMSC-Ex promoted wound healing and angiogenesis in vivo by using a rat skin burn model. We discovered that hucMSC-Ex promoted β-catenin nuclear translocation and induced the increased expression of proliferating cell nuclear antigen, cyclin D3, N-cadherin, and β-catenin and the decreased expression of E-cadherin. The activation of Wnt/β-catenin is critical in the induction of angiogenesis by hucMSC-Ex, which could be reversed by β-catenin inhibitor ICG-001. Wnt4 was delivered by hucMSC-Ex, and the knockdown of Wnt4 in hucMSC-Ex abrogated β-catenin nuclear translocation in endothelial cells. The in vivo proangiogenic effects were also inhibited by interference of Wnt4 expression in hucMSC-Ex. Taken together, these results suggest that hucMSC-Ex-mediated Wnt4 induces β-catenin activation in endothelial cells and exerts proangiogenic effects, which could be an important mechanism for cutaneous wound healing.
doi:10.5966/sctm.2014-0267
PMCID: PMC4414225  PMID: 25824139
Exosomes; Angiogenesis; Regenerative medicine; Wnt4; β-Catenin
3.  The Role of miR-34a in Tritiated Water Toxicity in Human Umbilical Vein Endothelial Cells 
Dose-Response  2016;14(2):1559325816638585.
In this work, we investigated the toxic effects of tritiated water (HTO) on the cardiovascular system. We examined the role of microRNA-34a (miR-34a) in DNA damage and repair in human umbilical vein endothelial cells (HUVECs) exposed to HTO. Cell proliferation capacity was evaluated by cell counting, and miR-34a expression was detected using quantitative PCR (QT-PCR). The Comet assay and γ-H2AX immunostaining were used to measure DNA double-strand breaks (DSBs). Reverse transcription polymerase chain reaction was used to measure the expression level of c-myc messenger RNA (mRNA). The cells exposed to HTO showed significantly lower proliferation than the control cells over 3 days. The DNA damage in the HTO group was more severe than that in the control group, at each time point examined. The expression of miR-34a mimics caused increased DNA DSBs whereas that of the miR-34a inhibitor caused decreased DNA DSBs. The proliferation viability was the opposite for the miR-34a mimics and inhibitor groups. The expression levels of c-myc mRNA in cells transfected with miR-34a mimics were lower than that in cells transfected with the miR-34a-5p inhibitor, at 0.5 hours and 2 hours after transfection. In summary, miR-34a mediates HTO toxicity in HUVECs by downregulating the expression of c-myc.
doi:10.1177/1559325816638585
PMCID: PMC4822198  PMID: 27099602
MiR-34a; tritiated water; DNA damage; c-myc
4.  Epigenetic modification of PKMζ rescues aging-related cognitive impairment 
Scientific Reports  2016;6:22096.
Cognition is impacted by aging. However, the mechanisms that underlie aging-associated cognitive impairment are unclear. Here we showed that cognitive decline in aged rats was associated with changes in DNA methylation of protein kinase Mζ (PKMζ) in the prelimbic cortex (PrL). PKMζ is a crucial molecule involved in the maintenance of long-term memory. Using different behavioral models, we confirmed that aged rats exhibited cognitive impairment in memory retention test 24 h after training, and overexpression of PKMζ in the PrL rescued cognitive impairment in aged rats. After fear conditioning, the protein levels of PKMζ and the membrane expression of GluR2 increased in the PrL in young and adult rats but not in aged rats, and the levels of methylated PKMζ DNA in the PrL decreased in all age groups, whereas the levels of unmethylated PKMζ DNA increased only in young and adult rats. We also found that environmentally enriched housing reversed the hypermethylation of PKMζ and restored cognitive performance in aged rats. Inactivation of PKMζ prevented the potentiating effects of environmental enrichment on memory retention in aged rats. These results indicated that PKMζ might be a potential target for the treatment of aging-related cognitive impairment, suggesting a potential therapeutic avenue.
doi:10.1038/srep22096
PMCID: PMC4772003  PMID: 26926225
5.  Tracking the Correlation Between CpG Island Methylator Phenotype and Other Molecular Features and Clinicopathological Features in Human Colorectal Cancers: A Systematic Review and Meta-Analysis 
Objectives:
The controversy of CpG island methylator phenotype (CIMP) in colorectal cancers (CRCs) persists, despite many studies that have been conducted on its correlation with molecular and clinicopathological features. To drive a more precise estimate of the strength of this postulated relationship, a meta-analysis was performed.
Methods:
A comprehensive search for studies reporting molecular and clinicopathological features of CRCs stratified by CIMP was performed within the PubMed, EMBASE, and Cochrane Library. CIMP was defined by either one of the three panels of gene-specific CIMP markers (Weisenberger panel, classic panel, or a mixture panel of the previous two) or the genome-wide DNA methylation profile. The associations of CIMP with outcome parameters were estimated using odds ratio (OR) or weighted mean difference (WMD) or hazard ratios (HRs) with 95% confidence interval (CI) for each study using a fixed effects or random effects model.
Results:
A total of 29 studies involving 9,393 CRC patients were included for analysis. We observed more BRAF mutations (OR 34.87; 95% CI, 22.49–54.06) and microsatellite instability (MSI) (OR 12.85 95% CI, 8.84–18.68) in CIMP-positive vs. -negative CRCs, whereas KRAS mutations were less frequent (OR 0.47; 95% CI, 0.30–0.75). Subgroup analysis showed that only the genome-wide methylation profile-defined CIMP subset encompassed all BRAF-mutated CRCs. As expected, CIMP-positive CRCs displayed significant associations with female (OR 0.64; 95% CI, 0.56–0.72), older age at diagnosis (WMD 2.77; 95% CI, 1.15–4.38), proximal location (OR 6.91; 95% CI, 5.17–9.23), mucinous histology (OR 3.81; 95% CI, 2.93–4.95), and poor differentiation (OR 4.22; 95% CI, 2.52–7.08). Although CIMP did not show a correlation with tumor stage (OR 1.10; 95% CI, 0.82–1.46), it was associated with shorter overall survival (HR 1.73; 95% CI, 1.27–2.37).
Conclusions:
The meta-analysis highlights that CIMP-positive CRCs take their own molecular feature, especially overlapping with BRAF mutations, and clinicopathological features and worse prognosis from CIMP-negative CRCs, suggesting CIMP could be used as an independent prognostic marker for CRCs.
doi:10.1038/ctg.2016.14
PMCID: PMC4822093  PMID: 26963001
6.  Synergistic Effect of Cold Atmospheric Plasma and Drug Loaded Core-shell Nanoparticles on Inhibiting Breast Cancer Cell Growth 
Scientific Reports  2016;6:21974.
Nano-based drug delivery devices allowing for effective and sustained targeted delivery of therapeutic agents to solid tumors have revolutionized cancer treatment. As an emerging biomedical technique, cold atmospheric plasma (CAP), an ionized non-thermal gas mixture composed of various reactive oxygen species, reactive nitrogen species, and UV photons, shows great potential for cancer treatment. Here we seek to develop a new dual cancer therapeutic method by integrating promising CAP and novel drug loaded core-shell nanoparticles and evaluate its underlying mechanism for targeted breast cancer treatment. For this purpose, core-shell nanoparticles were synthesized via co-axial electrospraying. Biocompatible poly (lactic-co-glycolic acid) was selected as the polymer shell to encapsulate anti-cancer therapeutics. Results demonstrated uniform size distribution and high drug encapsulation efficacy of the electrosprayed nanoparticles. Cell studies demonstrated the effectiveness of drug loaded nanoparticles and CAP for synergistic inhibition of breast cancer cell growth when compared to each treatment separately. Importantly, we found CAP induced down-regulation of metastasis related gene expression (VEGF, MTDH, MMP9, and MMP2) as well as facilitated drug loaded nanoparticle uptake which may aid in minimizing drug resistance-a major problem in chemotherapy. Thus, the integration of CAP and drug encapsulated nanoparticles provides a promising tool for the development of a new cancer treatment strategy.
doi:10.1038/srep21974
PMCID: PMC4768177  PMID: 26917087
7.  Development of a Novel Oral Cavity Compartmental Absorption and Transit Model for Sublingual Administration: Illustration with Zolpidem 
The AAPS Journal  2015;17(3):631-642.
Intraoral (IO) delivery is an alternative administration route to deliver a drug substance via the mouth that provides several advantages over conventional oral dosage forms. The purpose of this work was to develop and evaluate a novel, physiologically based oral cavity model for projection and mechanistic analysis of the clinical pharmacokinetics of intraoral formulations. The GastroPlus™ Oral Cavity Compartmental Absorption and Transit (OCCAT™) model was used to simulate the plasma concentration versus time profiles and the fraction and rate of intraoral drug transit/absorption for Intermezzo® sublingual tablets (zolpidem tartrate). The model was evaluated by the goodness-of-fit between simulated and observed concentrations and the deviation of key PK parameters (e.g., Cmax, Tmax, and AUC). In addition, a sensitivity analysis was conducted to demonstrate the interplay and impact of key modeling parameters on the fraction absorbed via oral mucosa (Fa_IO). The OCCAT™ model captured the observed pharmacokinetics for Intermezzo® sublingual tablets (R2 > 0.9). The predicted deviations (%) for Cmax, AUC0–inf, AUC0–20 min, and Tmax were 5.7, 28.0, 11.8, and 28.6%, respectively, indicating good prediction accuracy. The model also estimated ~18% of total drug was absorbed via the IO route. Furthermore, the sensitivity analysis indicated that the Fa_IO was not only associated with drug diffusivity and unbound fraction in epithelium tissue (fut) but also depended on the physicochemical properties of compounds for IO delivery (e.g., solubility and logDpH = 7.4). The novel physiologically based IO absorption OCCAT™ model showed satisfactory performance and will be helpful to guide development of future intraoral formulations.
Electronic supplementary material
The online version of this article (doi:10.1208/s12248-015-9727-7) contains supplementary material, which is available to authorized users.
doi:10.1208/s12248-015-9727-7
PMCID: PMC4406949  PMID: 25716146
diffusivity; intraoral delivery; oral cavity compartmental absorption and transit (OCCAT™) model; unbound fraction in epithelium tissue; zolpidem
8.  Difference in 24-Hour Urine Composition between Diabetic and Non-Diabetic Adults without Nephrolithiasis 
PLoS ONE  2016;11(2):e0150006.
Background
Diabetic patients are more likely to develop kidney stones than the general population. The underlying mechanisms for this disparity remain to be elucidated. Little is known about the relationship between urine composition and diabetes mellitus in non-stone-forming individuals. We sought to examine the differences in the 24-hour (24-h) urine composition between diabetic and non-diabetic adults who were not stone formers.
Methods
A convenience sample of 538 individuals without a history of nephrolithiasis, gout, hyperparathyroidism, or gastroenteric diseases participated in this study. The 24-h urine profiles of 115 diabetic adults were compared with those of 423 non-diabetic adults. Diabetes was defined by self-reported physician diagnosis or medication use. All participants were non-stone formers confirmed by urinary tract ultrasonography. Participants provided a fasting blood sample and a single 24-h urine collection for stone risk analysis. Student’s t-test was used to compare mean urinary values. Linear regression models were adjusted for age, gender, body mass index, hypertension, fasting serum glucose, serum total cholesterol, estimated creatinine clearance rate and urinary factors.
Results
Univariable analysis showed that the diabetic participants had significantly higher 24-h urine volumes and lower urine calcium and magnesium excretions than non-diabetic participants (all P < 0.05). After multivariate adjustment, no significant differences in 24-h urine composition were observed between diabetic and non-diabetic participants except for a slightly increased 24-h urine volume in diabetic participants (all P > 0.05). The main limitation of this study is that the convenience samples and self-reported data may have been sources of bias.
Conclusion
Our data showed that there were no differences in 24-h urine composition between diabetic and non-diabetic adults who are not stone formers. The reason for it might be the improved glycemic control in diabetic individuals in our study. Therefore, a tighter glycemic control might reduce stone formation in diabetic adults.
doi:10.1371/journal.pone.0150006
PMCID: PMC4764372  PMID: 26906900
9.  Impact of Mean Platelet Volume on Long-Term Mortality in Chinese Patients with ST-Elevation Myocardial Infarction 
Scientific Reports  2016;6:21350.
We investigated the association between mean platelet volume (MPV) and risk of all-cause mortality in Chinese patients with ST-Elevation Myocardial Infarction (STEMI). We enrolled 1836 patients with STEMI in Xuanwu Hospital from January 2008 to December 2013. Based on MPV, patients were categorized into the following groups: <9.5 fL (n = 85), 9.5–11.0 fL (n = 776), 11.1–12.5 fL (n = 811) and >12.5 fL (n = 164), respectively. Mean duration of follow-up was 56.9 months, and 197 patients (10.7%) died during follow-up. All-cause mortality rates were compared between groups. The lowest mortality occurred in patients with MPV between 9.5–11.0 fL, with a multivariable-adjusted hazard ratio (HR) of 1.15(95%CI 0.62–1.50), 1.38(95%CI 1.20–1.68), and 1.72(95%CI 1.41–1.96) in patients with MPV of <.5, 11.1–12.5 and >12.5 fL, respectively. Therefore, increased MPV was associated with all-cause mortality in Chinese patients with STEMI. MPV might be useful as a marker for risk stratification in Chinese patients with STEMI.
doi:10.1038/srep21350
PMCID: PMC4754908  PMID: 26879002
10.  Antibacterial Drug Leads: DNA and Enzyme Multi-Targeting 
Journal of medicinal chemistry  2015;58(3):1215-1227.
We report the results of an investigation of the activity of a series of amidine and bisamidine compounds against Staphylococcus aureus and Escherichia coli. The most active compounds bound to an AT-rich DNA dodecamer (CGCGAATTCGCG)2, and using DSC were found to increase the melting transition by up to 24 °C. Several compounds also inhibited undecaprenyl diphosphate synthase (UPPS) with IC50 values of 100–500 nM and we found good correlations (R2 = 0.89, S. aureus; R2 = 0.79, E. coli)) between experimental and predicted cell growth inhibition by using DNA ΔTm and UPPS IC50 experimental results together with 1 computed descriptor. We also solved the structures of three bisamidines binding to DNA as well as three UPPS structures. Overall, the results are of general interest in the context of the development of resistance-resistant antibiotics that involve multi-targeting.
doi:10.1021/jm501449u
PMCID: PMC4513954  PMID: 25574764
Drug discovery; Protein structure; DNA minor groove binder; Undecaprenyl diphosphate synthase
11.  Comparative studies of the composition of bacterial microbiota associated with the ruminal content, ruminal epithelium and in the faeces of lactating dairy cows 
Microbial Biotechnology  2016;9(2):257-268.
Summary
The objective of this research was to compare the composition of bacterial microbiota associated with the ruminal content (RC), ruminal epithelium (RE) and faeces of Holstein dairy cows. The RC, RE and faecal samples were collected from six Holstein dairy cows when the animals were slaughtered. Community compositions of bacterial 16S rRNA genes from RC, RE and faeces were determined using a MiSeq sequencing platform with bacterial‐targeting universal primers 338F and 806R. UniFrac analysis revealed that the bacterial communities of RC, RE and faeces were clearly separated from each other. Statistically significant dissimilarities were observed between RC and faeces (P = 0.002), between RC and RE (P = 0.003), and between RE and faeces (P = 0.001). A assignment of sequences to taxa showed that the abundance of the predominant phyla Bacteroidetes was lower in RE than in RC, while a significant higher (P < 0.01) abundance of Proteobacteria was present in RE than in RC. When compared with the RC, the abundance of Firmicutes and Verrucomicrobia was higher in faeces, and RC contained a greater abundance of Bacteroidetes and Tenericutes. A higher proportions of Butyrivibrio and Campylobacter dominated RE as compared to RC. The faecal microbiota was less diverse than RC and dominated by genera Turicibacter and Clostridium. In general, these findings clearly demonstrated the striking compositional differences among RC, RE and faeces, indicating that bacterial communities are specific and adapted to the harbouring environment.
doi:10.1111/1751-7915.12345
PMCID: PMC4767291  PMID: 26833450
12.  Network-Based Meta-Analyses of Associations of Multiple Gene Expression Profiles with Bone Mineral Density Variations in Women 
PLoS ONE  2016;11(1):e0147475.
Background
Existing microarray studies of bone mineral density (BMD) have been critical for understanding the pathophysiology of osteoporosis, and have identified a number of candidate genes. However, these studies were limited by their relatively small sample sizes and were usually analyzed individually. Here, we propose a novel network-based meta-analysis approach that combines data across six microarray studies to identify functional modules from human protein-protein interaction (PPI) data, and highlight several differentially expressed genes (DEGs) and a functional module that may play an important role in BMD regulation in women.
Methods
Expression profiling studies were identified by searching PubMed, Gene Expression Omnibus (GEO) and ArrayExpress. Two meta-analysis methods were applied across different gene expression profiling studies. The first, a nonparametric Fisher’s method, combined p-values from individual experiments to identify genes with large effect sizes. The second method combined effect sizes from individual datasets into a meta-effect size to gain a higher precision of effect size estimation across all datasets. Genes with Q test’s p-values < 0.05 or I2 values > 50% were assessed by a random effects model and the remainder by a fixed effects model. Using Fisher’s combined p-values, functional modules were identified through an integrated analysis of microarray data in the context of large protein–protein interaction (PPI) networks. Two previously published meta-analysis studies of genome-wide association (GWA) datasets were used to determine whether these module genes were genetically associated with BMD. Pathway enrichment analysis was performed with a hypergeometric test.
Results
Six gene expression datasets were identified, which included a total of 249 (129 high BMD and 120 low BMD) female subjects. Using a network-based meta-analysis, a consensus module containing 58 genes (nodes) and 83 edges was detected. Pathway enrichment analysis of the 58 module genes revealed that these genes were enriched in several important KEGG pathways including Osteoclast differentiation, B cell receptor signaling pathway, MAPK signaling pathway, Chemokine signaling pathway and Insulin signaling pathway. The importance of module genes was replicated by demonstrating that most module genes were genetically associated with BMD in the GWAS data sets. Meta-analyses were performed at the individual gene level by combining p-values and effect sizes. Five candidate genes (ESR1, MAP3K3, PYGM, RAC1 and SYK) were identified based on gene expression meta-analysis, and their associations with BMD were also replicated by two BMD meta-analysis studies.
Conclusions
In summary, our network-based meta-analysis not only identified important differentially expressed genes but also discovered biologically meaningful functional modules for BMD determination. Our study may provide novel therapeutic targets for osteoporosis in women.
doi:10.1371/journal.pone.0147475
PMCID: PMC4726665  PMID: 26808152
13.  MicroRNA-26a suppresses epithelial-mesenchymal transition in human hepatocellular carcinoma by repressing enhancer of zeste homolog 2 
Background
Our previous study reported that microRNA-26a (miR-26a) inhibited tumor progression by inhibiting tumor angiogenesis and intratumoral macrophage infiltration in hepatocellular carcinoma (HCC). The direct roles of miR-26a on tumor cell invasion remain poorly understood. In this study, we aim to explore the mechanism of miR-26a in modulating epithelial-mesenchymal transition (EMT) in HCC.
Methods
In vitro cell morphology and cell migration were compared between the hepatoma cell lines HCCLM3 and HepG2, which were established in the previous study. Overexpression and down-regulation of miR-26a were induced in these cell lines, and Western blot and immunofluorescence assays were used to detect the expression of EMT markers. Xenograft nude mouse models were used to observe tumor growth and pulmonary metastasis. Immunohistochemical assays were conducted to study the relationships between miR-26a expression and enhancer of zeste homolog 2 (EZH2) and E-cadherin expression in human HCC samples.
Results
Down-regulation of miR-26a in HCCLM3 and HepG2 cells resulted in an EMT-like cell morphology and high motility in vitro and increased in tumor growth and pulmonary metastasis in vivo. Through down-regulation of EZH2 expression and up-regulation of E-cadherin expression, miR-26a inhibited the EMT process in vitro and in vivo. Luciferase reporter assay showed that miR-26a directly interacted with EZH2 messenger RNA (mRNA). Furthermore, the expression of miR-26a was positively correlated with E-cadherin expression and inversely correlated with EZH2 expression in human HCC tissue.
Conclusions
miR-26a inhibited the EMT process in HCC by down-regulating EZH2 expression.
doi:10.1186/s13045-015-0229-y
PMCID: PMC4702409  PMID: 26733151
microRNA-26a (miR-26a); Hepatocellular carcinoma (HCC); Enhancer of zeste homolog 2 (EZH2); Epithelial-mesenchymal transition (EMT)
14.  Association of a Methylene Tetrahydrofolate Reductase C677T Polymorphism with Several Blood Chemical Levels in a Chinese Population 
Background: The methylene tetrahydrofolate reductase (MTHFR) C677T polymorphism is associated with hypertension in certain populations. This study investigated the relationship between the MTHFR polymorphism and hypertension and correlated blood lipid indexes, including homocysteine (HCY), lipoprotein (a) [Lp (a)], high-density lipoprotein (HDL), low-density lipoprotein (LDL), apolipoprotein A I (Apo AI), Apo B, glucose (GLU), total cholesterol (TC), and triglyceride (TG), in a Chinese population. Materials and Methods: A total of 174 patients with hypertension and 634 healthy control individuals from Jiangxi Province were recruited between June 2012 and September 2012 for genotyping of the MTHFR C677T polymorphism using polymerase chain reaction-restriction fragment length polymorphism. Biochemical parameters were also assessed in these subjects and statistically compared to the MTHFR C677T polymorphism and the risk for hypertension. Results: HCY and Lp (a) levels were significantly higher in subjects with a MTHFR 677TT genotype than in those with a CC/CT genotype, independent of hypertension. The frequency of the TT genotype and the T allele in hypertension patients was significantly higher than in the healthy controls. Furthermore, in the male hypertension patient group, the average levels of HCY, HDL, Apo AI, and TC were significantly different from those in female hypertension patients (pHCY=0.001, pHDL=0.004, pApo AI<0.001, pTC=0.012). In the male control group, the average levels of HCY, HDL, Apo AI, GLU, and TC were significantly different from those of female controls (pHCY<0.001, pHDL<0.001, pApo AI<0.001, pGLU=0.001, and pTC=0.004). Conclusion: Our data demonstrate that the MTHFR C677T polymorphism is positively correlated with an increased risk of hypertension through an increase in HCY levels. The blood lipid correlative index was different between male and female hypertension patients and controls.
doi:10.1089/gtmb.2014.0213
PMCID: PMC4278168  PMID: 25489783
15.  Development of Novel Three-Dimensional Printed Scaffolds for Osteochondral Regeneration 
Tissue Engineering. Part A  2014;21(1-2):403-415.
As modern medicine advances, various methodologies are being explored and developed in order to treat severe osteochondral defects in joints. However, it is still very challenging to cure the osteochondral defects due to their poor inherent regenerative capacity, complex stratified architecture, and disparate biomechanical properties. The objective of this study is to create novel three-dimensional (3D) printed osteochondral scaffolds with both excellent interfacial mechanical properties and biocompatibility for facilitating human bone marrow mesenchymal stem cell (MSC) growth and chondrogenic differentiation. For this purpose, we designed and 3D printed a series of innovative bi-phasic 3D models that mimic the osteochondral region of articulate joints. Our mechanical testing results showed that our bi-phasic scaffolds with key structures have enhanced mechanical characteristics in compression (a maximum Young's modulus of 31 MPa) and shear (a maximum fracture strength of 5768 N/mm2) when compared with homogenous designs. These results are also correlated with numerical simulation. In order to improve their biocompatibility, the scaffolds' surfaces were further modified with acetylated collagen (one of the main components in osteochondral extracellular matrix). MSC proliferation results demonstrated that incorporation of a collagen, along with biomimetically designed micro-features, can greatly enhance MSC growth after 5 days in vitro. Two weeks' chondrogenic differentiation results showed that our novel scaffolds (dubbed “key” scaffolds), both with and without surface collagen modification, displayed enhanced chondrogenesis (e.g., 130%, 114%, and 236% increases in glycosaminoglycan, type II collagen deposition, and total protein content on collagen-modified key scaffolds when compared with homogeneous controls).
doi:10.1089/ten.tea.2014.0138
PMCID: PMC4292864  PMID: 25088966
16.  DNA Methylation in the Exon 1 Region and Complex Regulation of Twist1 Expression in Gastric Cancer Cells 
PLoS ONE  2015;10(12):e0145630.
Twist1 overexpression is frequently observed in various cancers including gastric cancer (GC). Although DNA methylation of the Twist1 gene has been reported in cancer cells, the mechanisms underlying transcriptional activation remain uncertain. In this study, we first examined epigenetic alterations of the Twist1 using Twist1 transcription-positive and -negative cell lines that are derived from our established diffuse-type GC mouse model. Treatment with a DNA demethylation agent 5-aza-dC re-activated Twist1 expression in Twist1 expression-negative GC cells. According to methylation-specific PCR and bisulfite sequencing analysis, methylation at the CpG-rich region within Twist1 coding exon 1, rather than its promoter region, was tightly linked to transcriptional silencing of the Twist1 expression in mouse GC cells. Chromatin immunoprecipitation assays revealed that active histone mark H3K4me3 was enriched in Twist1 expression-positive cells, and inactive histone mark H3K9me3 was enriched in Twist1 expression-negative cells. The expression levels of Suv39h1 and Suv39h2, histone methyltransferases for H3K9me3, were inversely correlated with Twist1 expression, and knockdown of Suv39h1 or Suv39h2 induced Twist1 expression. Moreover, Sp1 transcription factor bound to the exon 1 CpG-rich region in Twist1 expression-positive cell lines, and Twist1 expression was diminished by mithramycin, which that interferes with Sp1 binding to CpG-rich regulatory sequences. Our studies suggested that the Twist1 transcription in GC cells might be regulated through potential cooperation of DNA methylation, histone modification in complex with Sp1 binding to CpG-rich regions within the exon 1 region.
doi:10.1371/journal.pone.0145630
PMCID: PMC4687923  PMID: 26695186
17.  Characterization and bioactivity of novel calcium antagonists - N-methoxy-benzyl haloperidol quaternary ammonium salt 
Oncotarget  2015;6(41):43759-43769.
BACKGROUND AND PURPOSE
Calcium antagonists play an important role in clinical practice. However, most of them have serious side effects. We have synthesized a series of novel calcium antagonists, quaternary ammonium salt derivatives of haloperidol with N-p-methoxybenzyl (X1), N-m-methoxybenzyl (X2) and N-o-methoxybenzyl (X3) groups. The objective of this study was to investigate the bioactivity of these novel calcium antagonists, especially the vasodilation activity and cardiac side-effects. The possible working mechanisms of these haloperidol derivatives were also explored.
EXPERIMENTAL APPROACH
Novel calcium antagonists were synthesized by amination. Compounds were screened for their activity of vasodilation on isolated thoracic aortic ring of rats. Their cardiac side effects were explored. The patch-clamp, confocal laser microscopy and the computer-fitting molecular docking experiments were employed to investigate the possible working mechanisms of these calcium antagonists.
RESULTS
The novel calcium antagonists, X1, X2 and X3 showed stronger vasodilation effect and less cardiac side effect than that of classical calcium antagonists. They blocked L-type calcium channels with an potent effect order of X1 > X2 > X3. Consistently, X1, X2 and X3 interacted with different regions of Ca2+-CaM-CaV1.2 with an affinity order of X1 > X2 > X3.
CONCLUSIONS
The new halopedidol derivatives X1, X2 and X3 are novel calcium antagonists with stronger vasodilation effect and less cardiac side effect. They could have wide clinical application.
PMCID: PMC4791264  PMID: 26544729
calcium; novel calcium antagonists; KCl-induced aortic ring contraction; synthesis
18.  Role of Penicillium chrysogenum XJ-1 in the Detoxification and Bioremediation of Cadmium 
Microbial bioremediation is a promising technology to treat heavy metal-contaminated soils. However, the efficiency of filamentous fungi as bioremediation agents remains unknown, and the detoxification mechanism of heavy metals by filamentous fungi remains unclear. Therefore, in this study, we investigated the cell morphology and antioxidant systems of Penicillium chrysogenum XJ-1 in response to different cadmium (Cd) concentrations (0–10 mM) by using physico-chemical and biochemical methods. Cd in XJ-1 was mainly bound to the cell wall. The malondialdehyde level in XJ-1 cells was increased by 14.82–94.67 times with the increase in Cd concentration. The activities of superoxide dismutase, glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G6PDH) peaked at 1 mM Cd, whereas that of catalase peaked at 5 mM Cd. Cd exposure increased the glutathione/oxidized glutathione ratio and the activities of GR and G6PDH in XJ-1. These results suggested that the Cd detoxification mechanism of XJ-1 included biosorption, cellular sequestration, and antioxidant defense. The application of XJ-1 in Cd-polluted soils (5–50 mg kg-1) successfully reduced bioavailable Cd and increased the plant yield, indicating that this fungus was a promising candidate for in situ bioremediation of Cd-polluted soil.
doi:10.3389/fmicb.2015.01422
PMCID: PMC4685053  PMID: 26733967
Penicillium chrysogenum; cadmium; antioxidant system; detoxification mechanism; bioremediation
19.  Reduction of Leukocyte Counts by Hydroxyurea Improves Cardiac Function in Rats with Acute Myocardial Infarction 
Background
This study aimed to decrease leukocytes counts by hydroxyurea (Hu) in an acute myocardial infarction (AMI) rat model and examine its effect on the inflammatory response of myocardial infarction and cardiac functions.
Material/Methods
AMI was successfully caused in 36 rats, and 12 control rats received sham operation. Rats in the AMI group were then randomly divided into Hu and vehicle group with 18 rats each. Rats in the Hu AMI group received Hu (200 mg/kg) intragastrically while vehicle AMI group received saline. Leukocytes counts, cardiac functions, myocardial tissue morphology, and levels of soluble intercellular adhesion molecule-1 (sICAM), P-selectin and platelet activating factor (PAF) were measured and compared among the three groups four weeks after AMI induction.
Results
Leukocytes, neutrophils, and leukomonocyte counts in vehicle AMI rats were significantly higher than that of the normal control group (p<0.05). However, Hu treatment decreased their counts significantly (p<0.05). sICAM, P-selectin, and PAF level in vehicle AMI group were significantly higher than those of the normal group, and their level was also decreased by Hu treatment (p<0.05). Echocardiography analysis showed that Hu treatment increased left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) compared to that of vehicle AMI group (p<0.05). Histopathological examination showed that Hu significantly reduced the swelling of the heart muscle fiber in necrotic foci and the number of inflammatory cells infiltrated into myocardial interstitium compared to vehicle AMI group.
Conclusions
Decrease leukocytes counts by Hu significantly reduced inflammatory reaction and improved cardiac functions in AMI rats.
doi:10.12659/MSM.893744
PMCID: PMC4687945  PMID: 26675565
Leukocyte Count; Myocardial Infarction; Rats, Wistar
20.  HERC5 is a prognostic biomarker for post-liver transplant recurrent human hepatocellular carcinoma 
Background and aims
Orthotopic liver transplantation (OLT) can be an effective treatment option for certain patients with early stage hepatocellular carcinoma (HCC) meeting Milan, UCSF, or Hangzhou criteria. However, HCC recurrence rates post-OLT range from 20 to 40 %, with limited follow-up options. Elucidating genetic drivers common to primary and post-OLT recurrent tumors may further our understanding and help identify predictive biomarkers of recurrence—both to ultimately help manage clinical decisions for patients undergoing OLT.
Methods
Whole exome and RNA sequencing in matched primary and recurrent tumors, normal adjacent tissues, and blood from four Chinese HCC patients was conducted. SiRNA knockdown and both qRT-PCR and Western assays were performed on PLCPRF5, SNU449 and HEPG2 cell lines; immunohistochemistry and RNA Sequencing were conducted on the primary tumors of Chinese HCC patients who experienced tumor recurrence post-OLT (n = 9) or did not experience tumor recurrence (n = 12).
Results
In three independent HCC studies of patients undergoing transplantation (n = 21) or surgical resection (n = 242, n = 44) of primary tumors (total n = 307), HERC5 mRNA under-expression correlated with shorter: time to tumor recurrence (p = 0.007 and 0.02) and overall survival (p = 0.0063 and 0.023), even after adjustment for relevant clinical variables. HERC5 loss drives CCL20 mRNA and protein over-expression and associates with regulatory T cell infiltration as measured by FOXP3 expression. Further, matched primary and recurrent tumors from the 4 HCC patients indicated clonal selection advantage of Wnt signaling activation and CDKN2A inactivation.
Conclusions
HERC5 plays a crucial role in HCC immune evasion and has clinical relevance as a reproducible prognostic marker for risk of tumor recurrence and survival in patients.
Electronic supplementary material
The online version of this article (doi:10.1186/s12967-015-0743-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s12967-015-0743-2
PMCID: PMC4676172  PMID: 26653219
Hepatocellular carcinoma; Orthotopic liver transplantation; Whole exome; HERC5
21.  Enhanced human bone marrow mesenchymal stem cell functions on cathodic arc plasma-treated titanium 
Surface modification of titanium for use in orthopedics has been explored for years; however, an ideal method of integrating titanium with native bone is still required to this day. Since human bone cells directly interact with nanostructured extracellular matrices, one of the most promising methods of improving titanium’s osseointegration involves inducing bio-mimetic nanotopography to enhance cell–implant interaction. In this regard, we explored an approach to functionalize the surface of titanium by depositing a thin film of textured titanium nanoparticles via a cathodic arc discharge plasma. The aim is to improve human bone marrow mesenchymal stem cell (MSC) attachment and differentiation and to reduce deleterious effects of more complex surface modification methods. Surface functionalization was analyzed by scanning electron microscopy, atomic force microscopy, contact angle testing, and specific protein adsorption. Scanning electron microscopy and atomic force microscopy examination demonstrate the deposition of titanium nanoparticles and the surface roughness change after coating. The specific fibronectin adsorption was enhanced on the modified titanium surface that associates with the improved hydrophilicity. MSC adhesion and proliferation were significantly promoted on the nanocoated surface. More importantly, compared to bare titanium, greater production of total protein, deposition of calcium mineral, and synthesis of alkaline phosphatase were observed from MSCs on nanocoated titanium after 21 days. The method described herein presents a promising alternative method for inducing more cell favorable nanosurface for improved orthopedic applications.
doi:10.2147/IJN.S92733
PMCID: PMC4677661  PMID: 26677327
titanium; mesenchymal stem cell; plasma; nanocoating; orthopedics
23.  Three-dimensional direct cell patterning in collagen hydrogels with near-infrared femtosecond laser 
Scientific Reports  2015;5:17203.
We report a methodology for three-dimensional (3D) cell patterning in a hydrogel in situ. Gold nanorods within a cell-encapsulating collagen hydrogel absorb a focused near-infrared femtosecond laser beam, locally denaturing the collagen and forming channels, into which cells migrate, proliferate, and align in 3D. Importantly, pattern resolution is tunable based on writing speed and laser power, and high cell viability (>90%) is achieved using higher writing speeds and lower laser intensities. Overall, this patterning technique presents a flexible direct-write method that is applicable in tissue engineering systems where 3D alignment is critical (such as vascular, neural, cardiac, and muscle tissue).
doi:10.1038/srep17203
PMCID: PMC4658636  PMID: 26603915
24.  Structural and Functional Analysis of Bacillus subtilis YisP Reveal a Role of its Product in Biofilm Production 
Chemistry & biology  2014;21(11):1557-1563.
Summary
YisP is involved in biofilm formation in Bacillus subtilis and has been predicted to produce C30 isoprenoids. We determined the structure of YisP and observe that it adopts the same fold as squalene and dehydrosqualene synthases. However, the first aspartate-rich motif found in essentially all isoprenoid synthases is aspartate-poor in YisP, and cannot catalyze head-to-head condensation reactions. We find that YisP acts as a phosphatase, catalyzing formation of farnesol from farnesyl diphosphate, and is the first phosphatase to adopt the fold seen in the head-to-head prenyl synthases. Farnesol restores biofilm formation in a Δyisp mutant, and modifies lipid membrane structure similarly to the virulence factor, staphyloxanthin. The work clarifies the role of YisP in biofilm formation and suggests an intriguing possibility that many of the YisP-like homologs found in other bacteria may also have interesting products and functions.
doi:10.1016/j.chembiol.2014.08.018
PMCID: PMC4252625  PMID: 25308276
25.  A combination therapy for KRAS-driven lung adenocarcinomas using lipophilic bisphosphonates and rapamycin 
Science translational medicine  2014;6(263):263ra161.
Lung cancer is the most common human malignancy and leads to about one-third of all cancer-related deaths. Lung adenocarcinomas harboring KRAS mutations, in contrast to those with EGFR and EML4-ALK mutations, have not yet been successfully targeted. Here, we describe a combination therapy for treating these malignancies using two agents: a lipophilic bisphosphonate and rapamycin. This drug combination is much more effective than either agent acting alone in the KRAS G12D induced mouse lung model. Lipophilic bisphosphonates inhibit both farnesyl and geranylgeranyldiphosphate synthases, effectively blocking prenylation of the KRAS and other small G-proteins critical for tumor growth and cell survival. Bisphosphonate treatment of cells initiated autophagy but was ultimately unsuccessful and led to p62 accumulation and concomitant NF-κB activation, resulting in dampened efficacy in vivo. However, we found that rapamycin, in addition to inhibiting the mTOR pathway, facilitated autophagy and prevented p62 accumulation-induced NF-κB activation and tumor cell proliferation. Overall, these results suggest that using lipophilic bisphosphonates in combination with rapamycin may provide an effective strategy for targeting lung adenocarcinomas harboring KRAS mutations.
doi:10.1126/scitranslmed.3010382
PMCID: PMC4326221  PMID: 25411474

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