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1.  Study on the Regulatory Mechanism of the Lipid Metabolism Pathways during Chicken Male Germ Cell Differentiation Based on RNA-Seq 
PLoS ONE  2015;10(2):e0109469.
Here, we explore the regulatory mechanism of lipid metabolic signaling pathways and related genes during differentiation of male germ cells in chickens, with the hope that better understanding of these pathways may improve in vitro induction. Fluorescence-activated cell sorting was used to obtain highly purified cultures of embryonic stem cells (ESCs), primitive germ cells (PGCs), and spermatogonial stem cells (SSCs). The total RNA was then extracted from each type of cell. High-throughput analysis methods (RNA-seq) were used to sequence the transcriptome of these cells. Gene Ontology (GO) analysis and the KEGG database were used to identify lipid metabolism pathways and related genes. Retinoic acid (RA), the end-product of the retinol metabolism pathway, induced in vitro differentiation of ESC into male germ cells. Quantitative real-time PCR (qRT-PCR) was used to detect changes in the expression of the genes involved in the retinol metabolic pathways. From the results of RNA-seq and the database analyses, we concluded that there are 328 genes in 27 lipid metabolic pathways continuously involved in lipid metabolism during the differentiation of ESC into SSC in vivo, including retinol metabolism. Alcohol dehydrogenase 5 (ADH5) and aldehyde dehydrogenase 1 family member A1 (ALDH1A1) are involved in RA synthesis in the cell. ADH5 was specifically expressed in PGC in our experiments and aldehyde dehydrogenase 1 family member A1 (ALDH1A1) persistently increased throughout development. CYP26b1, a member of the cytochrome P450 superfamily, is involved in the degradation of RA. Expression of CYP26b1, in contrast, decreased throughout development. Exogenous RA in the culture medium induced differentiation of ESC to SSC-like cells. The expression patterns of ADH5, ALDH1A1, and CYP26b1 were consistent with RNA-seq results. We conclude that the retinol metabolism pathway plays an important role in the process of chicken male germ cell differentiation.
doi:10.1371/journal.pone.0109469
PMCID: PMC4320113  PMID: 25658587
2.  Immunomodulatory Effects of Polysaccharide from Marine Fungus Phoma herbarum YS4108 on T Cells and Dendritic Cells 
Mediators of Inflammation  2014;2014:738631.
YCP, as a kind of natural polysaccharides from the mycelium of marine filamentous fungus Phoma herbarum YS4108, has great antitumor potential via enhancement of host immune response, but little is known about the molecular mechanisms. In the present study, we mainly focused on the effects and mechanisms of YCP on the specific immunity mediated by dendritic cells (DCs) and T cells. T cell /DC activation-related factors including interferon- (IFN-) γ, interleukin-12 (IL-12), and IL-4 were examined with ELISA. Receptor knock-out mice and fluorescence-activated cell sorting are used to analyze the YCP-binding receptor of T cells and DCs. RT-PCR is utilized to measure MAGE-A3 for analyzing the tumor-specific killing effect. In our study, we demonstrated YCP can provide the second signal for T cell activation, proliferation, and IFN-γ production through binding to toll-like receptor- (TLR-) 2 and TLR-4. YCP could effectively promote IL-12 secretion and expression of markers (CD80, CD86, and MHC II) via TLR-4 on DCs. Antigen-specific immunity against mouse melanoma cells was strengthened through the activation of T cells and the enhancement of capacity of DCs by YCP. The data supported that YCP can exhibit specific immunomodulatory capacity mediated by T cells and DCs.
doi:10.1155/2014/738631
PMCID: PMC4267005  PMID: 25525304
3.  Solvent induced rapid modulation of micro/nano structures of metal carboxylates coordination polymers: mechanism and morphology dependent magnetism 
Scientific Reports  2014;4:6023.
Rational modulation of morphology is very important for functional coordination polymers (CPs) micro/nanostructures, and new strategies are still desired to achieve this challenging target. Herein, organic solvents have been established as the capping agents for rapid modulating the growth of metal-carboxylates CPs in organic solvent/water mixtures at ambient conditions. Co-3,5-pyridinedicarboxylate (pydc) CPs was studied here as the example. During the reaction, the organic solvents exhibited three types of modulation effect: anisotropic growth, anisotropic growth/formation of new crystalline phase and the formation of new crystalline phase solely, which was due to the variation of their binding ability with metal cations. The following study revealed that the binding ability was critically affected by their functional groups and molecular size. Moreover, their modulation effect could be finely tuned by changing volume ratios of solvent mixtures. Furthermore, they could be applied for modulating other metal-carboxylates CPs: Co-1,3,5-benzenetricarboxylic (BTC), Zn-pydc and Eu-pydc etc. Additionally, the as-prepared Co-pydc CPs showed a fascinating morphology-dependent antiferromagnetic behavior.
doi:10.1038/srep06023
PMCID: PMC4129412  PMID: 25113225
4.  Evolution of Oleosin in Land Plants 
PLoS ONE  2014;9(8):e103806.
Oleosins form a steric barrier surface on lipid droplets in cytoplasm, preventing them from contacting and coalescing with adjacent droplets. Oleosin genes have been detected in numerous plant species. However, the presence of oleosin genes in the most basally diverging lineage of land plants, liverworts, has not been reported previously. Thus we explored whether liverworts have an oleosin gene. In Marchantia polymorpha L., a thalloid liverwort, one predicted sequence was found that could encode oleosin, possessing the hallmark of oleosin, a proline knot (-PX5SPX3P-) motif. The phylogeny of the oleosin gene family in land plants was reconstructed based on both nucleotide and amino acid sequences of oleosins, from 31 representative species covering almost all the main lineages of land plants. Based on our phylogenetic trees, oleosin genes were classified into three groups: M-oleosins (defined here as a novel group distinct from the two previously known groups), low molecular weight isoform (L-oleosin), and high molecular weight isoform (H-oleosin), according to their amino-acid organization, phylogenetic relationships, expression tissues, and immunological characteristics. In liverworts, mosses, lycophytes, and gymnosperms, only M-oleosins have been described. In angiosperms, however, while this isoform remains and is highly expressed in the gametophyte pollen tube, two other isoforms also occur, L-oleosins and H-oleosins. Phylogenetic analyses suggest that the M-oleosin isoform is the precursor to the ancestor of L-oleosins and H-oleosins. The later two isoforms evolved by successive gene duplications in ancestral angiosperms. At the genomic level, most oleosins possess no introns. If introns are present, in both the L-isoform and the M-isoform a single intron inserts behind the central region, while in the H-isoform, a single intron is located at the 5′-terminus. This study fills a major gap in understanding functional gene evolution of oleosin in land plants, shedding new light on evolutionary transitions of lipid storage strategies.
doi:10.1371/journal.pone.0103806
PMCID: PMC4126676  PMID: 25105766
5.  Neoadjuvant Chemotherapy with FOLFOX4 Regimen to Treat Advanced Gastric Cancer Improves Survival without Increasing Adverse Events: A Retrospective Cohort Study from a Chinese Center 
The Scientific World Journal  2014;2014:418694.
Background/Aim. To evaluate the clinical efficacy of FOLFOX4 (5-fluomumcil/leucovorin combined and oxaliplatin) neoadjuvant chemotherapy for advanced gastric cancer (AGC). Patients and Methods. Fifty-eight AGC patients were enrolled in this retrospective cohort study, 23 in the neoadjuvant group and 35 in the adjuvant group. R0 resection, survival, and adverse events were compared. Results. The two groups were well-matched, with no significant differences in R0 resection rate (82.6% versus 82.0%) and number of lymph nodes dissection (16 (0–49) versus 13 (3–40)) between the two groups (P > 0.05). The number of lymph node metastases in the neoadjuvant group (3 (0–14)) was significantly fewer than that in the adjuvant group (6 (0–27)) (P = 0.04). The neoadjuvant group had significantly better median overall survival (29.0 versus 22.0 months) and 3-year survival rate (73.9% versus 40.0%) than the adjuvant group (P = 0.013). The positive expression rate of Ki-67 in the neoadjuvant group (40.0%, 8/20) was lower than that in the adjuvant group (74.2%, 23/31; P = 0.015). Conclusion. The FOLFOX4 neoadjuvant chemotherapy could improve survival without increasing adverse events in patients with AGC.
doi:10.1155/2014/418694
PMCID: PMC4127276  PMID: 25136668
6.  DrugMap Central: an on-line query and visualization tool to facilitate drug repositioning studies 
Bioinformatics  2013;29(14):1834-1836.
Summary: Systematic studies of drug repositioning require the integration of multi-level drug data, including basic chemical information (such as SMILES), drug targets, target-related signaling pathways, clinical trial information and Food and Drug Administration (FDA)-approval information, to predict new potential indications of existing drugs. Currently available databases, however, lack query support for multi-level drug information and thus are not designed to support drug repositioning studies. DrugMap Central (DMC), an online tool, is developed to help fill the gap. DMC enables the users to integrate, query, visualize, interrogate, and download multi-level data of known drugs or compounds quickly for drug repositioning studies all within one system.
Availability: DMC is accessible at http://r2d2drug.org/DMC.aspx.
Contact: STWong@tmhs.org
doi:10.1093/bioinformatics/btt279
PMCID: PMC3702253  PMID: 23681121
7.  Effect of Thickness of HA-Coating on Microporous Silk Scaffolds Using Alternate Soaking Technology 
BioMed Research International  2014;2014:637821.
Hydroxyapatite (HA) can be coated on various materials surface and has the function of osteogenicity. Microporous silk scaffold has excellent biocompatibility. In this study, alternate soaking technology was used to coat HA on microporous silk scaffolds. However, the cell proliferation was found to decrease with the increasing thickness (cycles of soaking) of HA-coating. This study aims to determine the best thickness (cycles of soaking) of HA-coating on microporous silk scaffolds. The SEM observation showed that group with one cycle of alternate soaking (1C-HA) has the most optimal porosity like non-HA-modified microporous silk scaffolds. The proliferation of osteoblasts has no significant difference between noncoated HA (N-HA) and 1C-HA groups, which are both significantly higher than those in two cycles of soaking (2C-HA) and three cycles of soaking (3C-HA) groups. The transcription levels of specific genes (runx2 and osteonectin) in osteoblasts of 1C-HA group were significantly higher than those of N-HA group. Moreover, the levels showed no significant difference among 1C-HA, 2C-HA, and 3C-HA groups. In conclusion, microporous silk scaffold with 1 cycle of HA-coating can combine the biocompatibility of silk and osteogenicity of HA.
doi:10.1155/2014/637821
PMCID: PMC4100396  PMID: 25093176
8.  Immobilized Lentivirus Vector on Chondroitin Sulfate-Hyaluronate Acid-Silk Fibroin Hybrid Scaffold for Tissue-Engineered Ligament-Bone Junction 
BioMed Research International  2014;2014:816979.
The lack of a fibrocartilage layer between graft and bone remains the leading cause of graft failure after anterior cruciate ligament (ACL) reconstruction. The objective of this study was to develop a gene-modified silk cable-reinforced chondroitin sulfate-hyaluronate acid-silk fibroin (CHS) hybrid scaffold for reconstructing the fibrocartilage layer. The scaffold was fabricated by lyophilizing the CHS mixture with braided silk cables. The scanning electronic microscopy (SEM) showed that microporous CHS sponges were formed around silk cables. Each end of scaffold was modified with lentiviral-mediated transforming growth factor-β3 (TGF-β3) gene. The cells on scaffold were transfected by bonded lentivirus. In vitro culture demonstrated that mesenchymal stem cells (MSCs) on scaffolds proliferated vigorously and produced abundant collagen. The transcription levels of cartilage-specific genes also increased with culture time. After 2 weeks, the MSCs were distributed uniformly throughout scaffold. Deposited collagen was also found to increase. The chondral differentiation of MSCs was verified by expressions of collagen II and TGF-β3 genes in mRNA and protein level. Histology also confirmed the production of cartilage extracellular matrix (ECM) components. The results demonstrated that gene-modified silk cable-reinforced CHS scaffold was capable of supporting cell proliferation and differentiation to reconstruct the cartilage layer of interface.
doi:10.1155/2014/816979
PMCID: PMC4075190  PMID: 25019087
9.  Tumor loci and their interactions on mouse chromosome 19 that contribute to testicular germ cell tumors 
BMC Genetics  2014;15:65.
Background
Complex genetic factors underlie testicular germ cell tumor (TGCT) development. One experimental approach to dissect the genetics of TGCT predisposition is to use chromosome substitution strains, such as the 129.MOLF-Chr 19 (M19). M19 carries chromosome (Chr) 19 from the MOLF whereas all other chromosomes are from the 129 strain. 71% of M19 males develop TGCTs in contrast to 5% in 129 strain. To identify and map tumor loci from M19 we generated congenic strains harboring MOLF chromosome 19 segments on 129 strain background and monitored their TGCT incidence.
Results
We found 3 congenic strains that each harbored tumor promoting loci that had high (14%-32%) whereas 2 other congenics had low (4%) TGCT incidences. To determine how multiple loci influence TGCT development, we created double and triple congenic strains. We found additive interactions were predominant when 2 loci were combined in double congenic strains. Surprisingly, we found an example where 2 loci, both which do not contribute significantly to TGCT, when combined in a double congenic strain resulted in greater than expected TGCT incidence (positive interaction). In an opposite example, when 2 loci with high TGCT incidences were combined, males of the double congenic showed lower than expected TGCT incidence (negative interaction). For the triple congenic strain, depending on the analysis, the overall TGCT incidence could be additive or could also be due to a positive interaction of one region with others. Additionally, we identified loci that promote bilateral tumors or testicular abnormalities.
Conclusions
The congenic strains each with their characteristic TGCT incidences, laterality of tumors and incidence of testicular abnormalities, are useful for identification of TGCT susceptibility modifier genes that map to Chr 19 and also for studies on the genetic and environmental causes of TGCT development. TGCTs are a consequence of aberrant germ cell and testis development. By defining predisposing loci and some of the locus interactions from M19, this study further advances our understanding of the complex genetics of TGCTs, which is the most common cancer in young human males.
doi:10.1186/1471-2156-15-65
PMCID: PMC4053281  PMID: 24886204
Congenic strain; Chromosome substitution strain; M19; Testicular germ cell tumor; Modifiers; Epistasis
10.  Identification of Prognosis-Relevant Subgroups in Patients with Chemoresistant Triple Negative Breast Cancer 
Purpose
Triple-negative breast cancer (TNBC) patients with residual disease after neoadjuvant chemotherapy generally have worse outcome; however, some patients with residual tumor after neoadjuvant chemotherapy do not relapse. We hypothesize that there are subgroups of chemoresistant TNBC patients with different prognosis.
Experimental Design
Forty-nine chemoresistant cases from 111 TNBC patients treated with neoadjuvant chemotherapy (M.D. Anderson Cancer Center) constituted the discovery cohort, 25 chemoresistant samples from 47 neoadjuvant chemotherapy-treated TNBC (The Methodist Hospital) chosen for validation. Extended validation was performed in 269 operable TNBC predicted to be chemoresistant by expression pattern from published data sets.
Results
We established a 7-gene prognostic signature using dChip and gene set enrichment analyses. In the independent validation cohort, the classifier predicted correctly with positive predictive value of 75.0% and negative predictive value (i.e., relapse-free survival [RFS]) of 76.9% at 3 years. Those predicted to relapse had a hazard ratio (HR) of 4.67 (95%CI, 1.27–17.15) for relapse in 3 years. In extended validation, patients predicted not to relapse exhibited 3-year RFS of 78.9%, while the 3-year RFS was 48.5% for patients predicted to relapse, with HR of 2.61 (95%CI, 1.52–4.49). The TNBC subgroup predicted to have relatively favorable prognosis was characterized by high expression of “luminal-like” genes (androgen-receptor [AR] and GATA3); while the subgroup with worse prognosis was characterized by expression of cancer stem-cell markers.
Conclusion
We developed a clinically relevant signature for patients with chemoresistant TNBC. For these women, new therapeutic strategies like targeting AR-activation or cancer stem-cells may need to be developed.
doi:10.1158/1078-0432.CCR-12-2986
PMCID: PMC3655097  PMID: 23549873
11.  Association of Plasma IL-6 and Hsp70 with HRV at Different Levels of PAHs Metabolites 
PLoS ONE  2014;9(4):e92964.
Background
Exposure to polycyclic aromatic hydrocarbons (PAHs) is associated with reduced heart rate variability (HRV), a strong predictor of cardiovascular diseases, but the mechanism is not well understood.
Objectives
We hypothesized that PAHs might induce systemic inflammation and stress response, contributing to altered cardiac autonomic function.
Methods
HRV indices were measured using a 3-channel digital Holter monitor in 800 coke oven workers. Plasma levels of interleukin-6 (IL-6) and heat shock protein 70 (Hsp70) were determined using ELISA. Twelve urinary PAHs metabolites (OH-PAHs) were measured by gas chromatography-mass spectrometry.
Results
We found that significant dose-dependent relationships between four urinary OH-PAHs and IL-6 (all Ptrend<0.05); and an increase in quartiles of IL-6 was significantly associated with a decrease in total power (TP) and low frequency (LF) (Ptrend = 0.014 and 0.006, respectively). In particular, elevated IL-6 was associated in a dose-dependent manner with decreased TP and LF in the high-PAHs metabolites groups (all Ptrend<0.05), but not in the low-PAHs metabolites groups. No significant association between Hsp70 and HRV in total population was found after multivariate adjustment. However, increased Hsp70 was significantly associated with elevated standard deviation of NN intervals (SDNN), TP and LF in the low-PAHs metabolites groups (all Ptrend<0.05). We also observed that both IL-6 and Hsp70 significantly interacted with multiple PAHs metabolites in relation to HRV.
Conclusions
In coke oven workers, increased IL-6 was associated with a dose-response decreased HRV in the high-PAHs metabolites groups, whereas increase of Hsp70 can result in significant dose-related increase in HRV in the low-PAHs metabolites groups.
doi:10.1371/journal.pone.0092964
PMCID: PMC3982957  PMID: 24722336
12.  Association of LOX-1 gene polymorphisms with cerebral infarction in northern Chinese Han population 
Background
Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) plays an important role in the pathophysiology of atherosclerosis and thrombosis. This study is aimed at evaluating the potential association of 3’-UTR-C188T and G501C in LOX-1 gene with cerebral infarction.
Methods
A total of 386 patients with cerebral infarction and 386 healthy controls were included in the study, which were unrelated Chinese Han population in the Liaoning Province of northern China. The single nucleotide polymorphisms, 3’-UTR-C188T and G501C, were analyzed by polymerase chain reaction–ligation detection reaction method.
Results
The frequencies of CC + GC genotype, GC genotype and C allele of G501C in the patients with cerebral infarction were significantly higher than those in the controls (P < 0.01, P < 0.01, P = 0.04, respectively). The correlation still remained after adjusting for confounding risk factors of cerebral infarction. In addition, no significant association was observed between 3’-UTR-C188T and cerebral infarction.
Conclusions
The study indicated that the G501C variant in LOX-1 gene may be associated with susceptibility to cerebral infarction, independent of other common risk factors, in northern Chinese Han population.
doi:10.1186/1476-511X-13-55
PMCID: PMC3994314  PMID: 24661313
LOX-1; Ox-LDL; Polymorphisms; Cerebral infarction; Atherosclerosis
13.  Chloroethylating and methylating dual function antineoplastic agents display superior cytotoxicity against repair proficient tumor cells 
Two new agents based upon the structure of the clinically active prodrug laromustine were synthesized. These agents, 2-(2-chloroethyl)-N-methyl-1,2-bis(methylsulfonyl)-N-nitrosohydrazinecarboxamide (1) and N-(2-chloroethyl)-2-methyl-1,2-bis(methylsulfonyl)-N-nitrosohydrazinecarboxamide (2), were designed to retain the potent chloroethylating and DNA cross-linking functions of laromustine, and gain the ability to methylate DNA at the O-6 position of guanine, while lacking the carbamoylating activity of laromustine. The methylating arm was introduced with the intent of depleting the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT). Compound 1 is markedly more cytotoxic than laromustine in both AGT minus EMT6 mouse mammary carcinoma cells and high AGT expressing DU145 human prostate carcinoma cells. DNA cross-linking studies indicated that its cross-linking efficiency is nearly identical to its predicted active decomposition product, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE), which is also produced by laromustine. AGT ablation studies in DU145 cells demonstrated that 1 can efficiently deplete AGT. Studies assaying methanol and 2-chloroethanol production as a consequence of the methylation and chloroethylation of water by 1 and 2 confirmed their ability to function as methylating and chloroethylating agents and provided insights into the superior activity of 1.
doi:10.1016/j.bmcl.2013.01.016
PMCID: PMC3594432  PMID: 23395657
chloroethylating; O6-alkylguanine-DNA alkyltransferase; 1, 2-bis(sulfonyl)hydrazines; methylating; laromustine; dual function
14.  A critical evaluation of random copolymer mimesis of homogeneous antimicrobial peptides 
Macromolecules  2013;46(5):1908-1915.
Polymeric synthetic mimics of antimicrobial peptides (SMAMPs) have recently demonstrated similar antimicrobial activity as natural antimicrobial peptides (AMPs) from innate immunity. This is surprising, since polymeric SMAMPs are heterogeneous in terms of chemical structure (random sequence) and conformation (random coil), in contrast to defined amino acid sequence and intrinsic secondary structure. To understand this better, we compare AMPs with a ‘minimal’ mimic, a well characterized family of polydisperse cationic methacrylate-based random copolymer SMAMPs. Specifically, we focus on a comparison between the quantifiable membrane curvature generating capacity, charge density, and hydrophobicity of the polymeric SMAMPs and AMPs. Synchrotron small angle x-ray scattering (SAXS) results indicate that typical AMPs and these methacrylate SMAMPs generate similar amounts of membrane negative Gaussian curvature (NGC), which is topologically necessary for a variety of membrane-destabilizing processes. Moreover, the curvature generating ability of SMAMPs is more tolerant of changes in the lipid composition than that of natural AMPs with similar chemical groups, consistent with the lower specificity of SMAMPs. We find that, although the amount of NGC generated by these SMAMPs and AMPs are similar, the SMAMPs require significantly higher levels of hydrophobicity and cationic charge to achieve the same level of membrane deformation. We propose an explanation for these differences, which has implications for new synthetic strategies aimed at improved mimesis of AMPs.
doi:10.1021/ma302577e
PMCID: PMC3671498  PMID: 23750051
15.  Population-Based Efficacy Modeling of Omalizumab in Patients with Severe Allergic Asthma Inadequately Controlled with Standard Therapy 
The AAPS Journal  2013;15(2):559-570.
Omalizumab, a recombinant humanized monoclonal antibody, is the first approved anti-immunoglobulin E (IgE) agent for the treatment of subjects with moderate to severe persistent allergic asthma that are inadequately controlled by the standard of care. The objective of this study was to quantitatively characterize relationships between serum free IgE and pulmonary function (as measured by forced expiratory volume in 1 s [FEV1]) as well as serum free IgE and airway inflammation (as measured by fractional exhaled nitric oxide [FeNO]) using population-based efficacy models. Data were collected from patients in the EXTRA trial who received omalizumab or placebo 150 to 375 mg subcutaneously every 2 or 4 weeks from week 0 to 48 with constant standard of care as background therapy. None of the covariates evaluated, including demographics, disease status, and baseline pharmacodynamic biomarkers, were significant in explaining the variability in the FEV1 or FeNO response to omalizumab. Results from the efficacy models further confirmed the current omalizumab dosing rationale based on the mean target free IgE level of 25 ng/ml and quantified the variability for the target. In addition, the resulting population models could be used to predict population FEV1 or FeNO response for omalizumab and/or other anti-IgE therapeutics for which PK-IgE models are constructed.
Electronic supplementary material
The online version of this article (doi:10.1208/s12248-013-9463-9) contains supplementary material, which is available to authorized users.
doi:10.1208/s12248-013-9463-9
PMCID: PMC3675747  PMID: 23413101
asthma; FEV1; IgE; omalizumab; population modeling
16.  Hypoxia-selective O6-Alkylguanine-DNA Alkyltransferase Inhibitors: Design, Synthesis and Evaluation of 6-(Benzyloxy)-2-(aryldiazenyl)-9H-purines as Prodrugs of O6-Benzylguanine 
Journal of medicinal chemistry  2013;56(3):1355-1359.
O6-Alkylguanine-DNA alkyltransferase (AGT) is a DNA repair protein which removes alkyl groups from the O-6 position of guanine, thereby providing strong resistance to anticancer agents which alkylate this position. The clinical usefulness of these anticancer agents would be substantially augmented if AGT could be selectively inhibited in tumor tissue, without a corresponding depletion in normal tissue. We report the synthesis of a new AGT inhibitor (5c) which selectively depletes AGT in hypoxic tumor cells.
doi:10.1021/jm301804p
PMCID: PMC3722860  PMID: 23311288
17.  Embryonic Trophoblasts Induce Decidual Regulatory T Cell Differentiation and Maternal–Fetal Tolerance through Thymic Stromal Lymphopoietin Instructing Dendritic Cells 
Physiological pregnancy requires the maternal immune system to recognize and tolerate embryonic Ags. Although multiple mechanisms have been proposed, it is not yet clear how the fetus evades the maternal immune system. In this article, we demonstrate that trophoblast-derived thymic stromal lymphopoietin (TSLP) instructs decidual CD11c+ dendritic cells (dDCs)with increased costimulatory molecules; MHC class II; and Th2/3-type, but not Th1-type, cytokines. TSLP-activated dDCs induce proliferation and differentiation of decidual CD4+CD25− T cells into CD4+CD25+FOXP3+ regulatory T cells (Tregs) through TGF-β1. TSLP-activated dDC–induced Tregs display immunosuppressive features and express Th2-type cytokines. In addition, decidual CD4+CD25+FOXP3+ Tregs promote invasiveness and HLA-G expression of trophoblasts, resulting in preferential production of Th2 cytokines and reduced cytotoxicity in decidual CD56brightCD16− NK cells. Of interest, decreased TSLP expression and reduced numbers of Tregs were observed at the maternal–fetal interface during miscarriage. Our study identifies a novel feedback loop between embryo-derived trophoblasts and maternal decidual leukocytes, which induces a tolerogenic immune response to ensure a successful pregnancy.
doi:10.4049/jimmunol.1203425
PMCID: PMC3918863  PMID: 24453244
18.  The Strain at Bone-Implant Interface Determines the Effect of Spinopelvic Reconstruction following Total Sacrectomy: A Strain Gauge Analysis in Various Spinopelvic Constructs 
PLoS ONE  2014;9(1):e85298.
Purpose
There is still some controversy regarding the optimal biomechanical concept for spinopelvic stabilization following total sacrectomy for malignancy. Strains at specific anatomical sites at pelvis/sacrum and implants interfaces have been poorly investigated. Herein, we compared and analyzed the strains applied at key points at the bone-implant interface in four different spinopelvic constructs following total sacrectomy; consequently, we defined a balanced architecture for spinopelvic fusion in that situation.
Methods
Six human cadaveric specimens, from second lumbar vertebra to proximal femur, were used to compare the partial strains at specific sites in a total sacrectomy model. Test constructs included: (1) intact pelvis (control), (2) sacral-rod reconstruction (SRR), (3) bilateral fibular flap reconstruction (BFFR), (4) four-rods reconstruction (FRR), and (5) improved compound reconstruction (ICR). Strains were measured by bonded strain gauges onto the surface of three specific sites (pubic rami, arcuate lines, and posterior spinal rods) under a 500 N axial load.
Results
ICR caused lower strains at specific sites and, moreover, on stress distribution and symmetry, compared to the other three constructs. Strains at pubic rami and arcuate lines following BFFR were lower than those following SRR, but higher at the posterior spinal rod construct. The different modes of strain distribution reflected different patient’s parameter-related conditions. FRR model showed the highest strains at all sites because of the lack of an anterior bracing frame.
Conclusions
The findings of this investigation suggest that both anterior bracing frame and the four-rods load dispersion provide significant load sharing. Additionally, these two constructs decrease the peak strains at bone-implant interface, thus determining the theoretical surgical technique to achieve optimal stress dispersion and balance for spinopelvic reconstruction in early postoperative period following total sacrectomy.
doi:10.1371/journal.pone.0085298
PMCID: PMC3891848  PMID: 24454839
19.  Empirical Evidence Supporting Frequent Cryptic Speciation in Epiphyllous Liverworts: A Case Study of the Cololejeunea lanciloba Complex 
PLoS ONE  2013;8(12):e84124.
Cryptic species are frequently recovered in plant lineages, and considered an important cause for divergent of morphological disparity and species diversity. The identification of cryptic species has important implications for the assessment of conservation needs of species aggregates. The mechanisms and processes of the origin of cryptic species diversity are still poorly understand based on the lack of studies especially in context of environment factors. Here we explored evidence for cryptic species within the epiphyllous liverworts Cololejeunea lanciloba complex based on two loci, the plastid trnL-F region and the nuclear ribosomal ITS region. Several analytic approaches were employed to delimit species based on DNA sequence variation including phylogenetic reconstruction, statistical parsimony networks analysis and two recently introduced species delimitation criteria: Rosenberg’s reciprocal monophyly and Rodrigo’s randomly distinct. We found evidence for thirteen genetically distinct putative species, each consisting of more than one haplotype, rather than four morphologically-circumscribed species. The results implied that the highly conserved phenotypes are not congruent with the genetic differentiation, contributing to incorrect assessments of the biodiversity of epiphyllous liverworts. We hypothesize that evolution of cryptic species recovered may be caused by selection of traits critical to the survival in epiphyllous habitats combined with limited developmental options designed in the small body.
doi:10.1371/journal.pone.0084124
PMCID: PMC3867491  PMID: 24367634
20.  Recurrent seminomas: Clinical features and biologic implications 
Urologic oncology  2010;30(4):10.1016/j.urolonc.2010.05.011.
Objectives
Certain patients with seminoma and clinically atypical phenotypes—visceral metastases, elevated levels of βhuman chorionic gonadotropin (βHCG), and/or recurrent disease— have a poor prognosis. The primary goal of this pilot study was to characterize the clinical characteristics and treatment profile of these rare patients. We also wished to test whether these tumors expressed any specific biomarkers that might distinguish them as a unique subtype of seminoma.
Materials and methods
We retrospectively identified 25 patients with a history of seminoma plus visceral metastases, βHCG levels >200 mU/ml, and/or recurrent disease. We reviewed these patients’ histories for treatment efficacy and clinical outcome. Tissue samples were available from 6 of those patients, and we studied them for expression of the markers OCT 3/4, PLAP, CD30, TRA-1-60, c-kit, and gp200. We compared our results with the expression of those markers in tissue samples from mixed seminoma/embryonal carcinomas and classic seminomas.
Results
Our analysis suggested that certain chemotherapeutic regimens (such as ifosfamide, paclitaxel, and cisplatin) are efficacious for the treatment of patients with these atypical seminomas. Further, specimens from the atypical seminomas generally had staining profiles that resembled those of classic seminomas and the seminoma components in mixed germ-cell tumors, but the profiles differed from those of the embryonal carcinoma components in the same mixed germ-cell tumors.
Conclusions
Although these atypical seminomas tend to be resistant to chemotherapy, they may still respond to certain chemotherapeutic regimens. Our pilot immunohistochemical study also suggested that the unique phenotypes associated with these atypical seminomas do not result from any relationship with embryonal carcinomas. More study is needed to confirm these initial findings.
doi:10.1016/j.urolonc.2010.05.011
PMCID: PMC3856193  PMID: 20822932
Seminoma; Testicular neoplasms; Embryonal carcinoma; TRA-1-60; gp200
21.  Design of a Hypoxia-Activated Prodrug Inhibitor of O6-Alkylguanine-DNA Alkyltransferase 
The efficacy of agents that alkylate the O-6 position of guanine is inhibited by O6-alkylguanine-DNA alkyltransferase (AGT) which removes these lesions from the tumor DNA. To increase differential toxicity, inhibitors must selectively deplete AGT in tumors, while sparing normal tissues where this protein serves a protective function. A newly synthesized prodrug of the AGT inhibitor O6-benzylguanine (O6-BG) with an α,α-dimethyl-4-nitrobenzyloxycarbonyl moiety masking the essential 2-amino group has demonstrated the feasibility of targeting hypoxic regions that are unique to solid tumors, for drug delivery. However, these modifications resulted in greatly decreased solubility. Recently, new potent global AGT inhibitors with improved formulatability such as O6-[(3-aminomethyl)benzylguanine (1) have been developed. However, acetylamino (N-(3-(((2-amino-9H-purin-6-yl)oxy)methyl)benzyl)acetamide) (2) exhibits a pronounced decrease in activity. Thus, 1 would be inactivated by N-acetylation and probably N-glucuronidation. To combat potential conjugational inactivation while retaining favorable solubility, we synthesized 6-((3-((dimethylamino)methyl)benzyl)oxy)-9H-purin-2-amine (3) in which the 3-aminomethyl moiety is protected by methylation; and to impart tumor selectivity we synthesized 2-(4-nitrophenyl)propan-2-yl(6-((3-((dimethylamino)methyl)benzyl)oxy)-9H-purin-2-yl)carbamate (7), a hypoxia targeted prodrug of 3 utilizing an α,α-dimethyl-4-nitrobenzyloxycarbonyl moiety. Consistent with this design, 7 demonstrates both hypoxia selective conversion by EMT6 cells of 7 to 3 and hypoxic sensitization of AGT containing DU145 cells to the cytotoxic actions of laromustine, while exhibiting improved solubility.
doi:10.1016/j.bmcl.2012.08.008
PMCID: PMC3444633  PMID: 22932317
22.  7-Nitro-4-(phenylthio)benzofurazan is a potent generator of superoxide and hydrogen peroxide 
Archives of toxicology  2012;86(10):1613-1625.
Here, we report on 7-nitro-4-(phenylthio) benzofurazan (NBF-SPh), the most potent derivative among a set of patented anticancer 7-nitrobenzofurazans (NBFs), which have been suggested to function by perturbing protein–protein interactions. We demonstrate that NBF-SPh participates in toxic redox-cycling, rapidly generating reactive oxygen species (ROS) in the presence of molecular oxygen, and this is the first report to detail ROS production for any of the anticancer NBFs. Oxygraph studies showed that NBF-SPh consumes molecular oxygen at a substantial rate, rivaling even plumbagin, menadione, and juglone. Biochemical and enzymatic assays identified superoxide and hydrogen peroxide as products of its redox-cycling activity, and the rapid rate of ROS production appears to be sufficient to account for some of the toxicity of NBF-SPh (LC50 = 12.1 µM), possibly explaining why tumor cells exhibit a sharp threshold for tolerating the compound. In cell cultures, lipid peroxidation was enhanced after treatment with NBF-SPh, as measured by 2-thiobarbituric acid-reactive substances, indicating a significant accumulation of ROS. Thioglycerol rescued cell death and increased survival by 15-fold to 20-fold, but pyruvate and uric acid were ineffective protectants. We also observed that the redox-cycling activity of NBF-SPh became exhausted after an average of approximately 19 cycles per NBF-SPh molecule. Electrochemical and computational analyses suggest that partial reduction of NBF-SPh enhances electrophilicity, which appears to encourage scavenging activity and contribute to electrophilic toxicity.
doi:10.1007/s00204-012-0872-9
PMCID: PMC3781597  PMID: 22669514
Benzofurazan; Reactive oxygen species; Oxidative stress; Electrochemistry; Electrophilic stress
23.  Hyaluronan-CD44 Interaction Promotes Growth of Decidual Stromal Cells in Human First-Trimester Pregnancy 
PLoS ONE  2013;8(9):e74812.
Hyaluronan (HA) and its receptor CD44 are expressed at the maternal-fetal interface, but its role in early pregnancy remains unclear. Here, we found that primary decidual stromal cells (DSCs) continuously secreted HA and expressed its receptor CD44. Pregnancy-associated hormones up-regulated HA synthetase (HAS) 2 transcription and HA release from DSCs. High molecular weight-HA (HMW-HA), but not medium molecular weight (MMW-HA) or low molecular weight (LMW-HA), promoted proliferation and inhibited apoptosis of DSCs in a CD44-dependent manner. The in-cell Western analysis revealed HMW-HA activated PI3K/AKT and mitogen-activated protein kinase (MAPK)/ERK1/2 signaling pathways time-dependently. Blocking these pathways by specific inhibitor LY294002 or U0126 abrogated HMW-HA-regulated DSc proliferation and apoptosis. Finally, we have found that HA content, HA molecular weight, HAS2 mRNA level, and CD44 expression were significantly decreased in DSCs from unexplained miscarriage compared with the normal pregnancy. Collectively, our results indicate that higher level and greater molecular mass of HA at maternal-fetal interface contributes to DSc growth and maintenance of DSCs in human early pregnancy.
doi:10.1371/journal.pone.0074812
PMCID: PMC3777984  PMID: 24069351
24.  Cyclosporin A promotes proliferating cell nuclear antigen expression and migration of human cytotrophoblast cells via the mitgen-activated protein kinase-3/1-mediated nuclear factor-κB signaling pathways 
Our previous studies have demonstrated that cyclosporin A (CsA) promotes the proliferation and migration of human trophoblasts via the mitgen-activated protein kinase-3/1 (MAPK3/1) pathway. In the present study, we further investigated the role of nuclear factor (NF)-κB in the CsA-induced trophoblast proliferating cell nuclear antigen (PCNA) expression and migration, and its relationship to MAPK3/1 signal. Flow cytometry was used to analyze the expression of PCNA in trophoblasts. The migration of human primary trophoblasts was determined by wound-healing assay and transwell migration assay. Western blot analysis was performed to evaluate the activation of NF-κB p65 and NF-κB inhibitory protein I-κB in human trophoblasts. We found that treatment with CsA promotes PCNA expression and migration of human trophoblast in a dose-associated manner. Blocking of the MAPK3/1 signal abrogated the enhanced PCNA expression and migration in trophoblasts by CsA. In addition, CsA increased the phosphorylation of NF-κB p65 and the inhibitor I-κB in human trophoblasts in a time-related manner. Pretreatment with MAPK3/1 inhibitor U0126 abrogated the phosphorylation of NF-κB p65 and I-κB. Accordingly, the CsA-induced enhancement of PCNA expression and migration in trophoblasts was also decreased. This CsA-induced enhancement in the expression and migration of trophoblasts was abolished by pretreatment with pyrrolidine dithiocarbamate, a specific NF-κB inhibitor. Thus, our results suggest that CsA promotes PCNA expression and migration of human trophoblasts via MAPK-mediated NF-κB activation.
PMCID: PMC3796221  PMID: 24133577
Cyclosporine A; trophoblast; PCNA; migration; signal transduction pathway
25.  Isoniazid pharmacokinetics, pharmacodynamics and dosing in South African infants 
Therapeutic Drug Monitoring  2012;34(4):446-451.
Aims
There are limited data on isoniazid (INH) pharmacokinetics in infants and young children and, therefore, uncertainty on appropriate dosing.
Methods
Pharmacokinetic data were obtained from perinatally HIV-exposed South African infants ages 3–24 months receiving INH 10–20 mg/kg/day orally for Mycobacterium tuberculosis (TB) prophylaxis. INH pharmacokinetic parameters were characterized with a population pharmacokinetic approach. Dosing simulations were performed to evaluate weight-based INH doses in children based on N-acetyltransferase 2 enzyme (NAT2) genotype, age, maximum concentrations (Cmax) ≥ 3mg/L, and area under the curve (AUC0-24) ≥ 10.52 mg*hr/L.
Results
In 151 infants (53% female, 48% HIV positive) receiving a mean INH dose of 14.5 mg/kg/day, mean (±SD) Cmax at 3, 6, and 23 months of age were 10.0 (3.5), 8.6 (2.6), and 9.3 (3.8) mg/L, respectively, mean (±SD) AUC0-24 were 53.6 (26.8), 42 (19.9), and 44 (30.7) mg*hr/L, respectively, and mean (±SD) half-life were 2.1 (0.7), 1.9 (0.6), and 1.8 (0.9) hours, respectively. A trimodal apparent oral clearance of INH as a function of NAT2 genotype was apparent as early as 3 months. INH was well tolerated. At an average INH dose of 14.5 mg/kg/day, 99% of infants ages 3–24 months have an INH Cmax ≥ 3 mg/L and 98% have an INH AUC0-24 ≥ 10.52 mg*hr/L.
Conclusions
INH at an average dose of 14.5 mg/kg once daily was well tolerated in infants and achieved INH Cmax values ≥ 3 mg/L and AUC0-24 values ≥ 10.52 mg*hr/L.
doi:10.1097/FTD.0b013e31825c4bc3
PMCID: PMC3397663  PMID: 22695364
isoniazid; pharmacokinetics; dosing; infants; children

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