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1.  Role of the ER/NO/cGMP Signaling Pathway in the Promotion of Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells by Actaea racemosa Extract 
Purpose/Objective. To investigate the effect of Actaea racemosa (AR) extract on in vitro osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) via the ER/NO/cGMP signaling pathway. Methods/Materials. Rat BMSCs were treated with osteogenic differentiation-inducing medium containing AR; estrogen receptor antagonist, ICI 182,780 (10−6 mol/L); and nitric oxide synthase inhibitor, L-nitro arginine methyl ester (L-NAME, 6 × 10−3 mol/L). Markers of osteogenic differentiation (alkaline phosphatase [ALP] activity, osteocalcin secretion, and calcium ion deposit levels) and the levels of key signaling molecules (nitric oxide synthase [NOS], nitric oxide [NO], and cyclic guanosine monophosphate [cGMP]) were assessed. Results. AR (10−1–10−6 g/L) increased ALP activity in a dose-dependent manner, and the highest ALP, osteocalcin, and osteoprotegerin activities were achieved at an AR concentration of 10−4 g/L. Therefore, the concentration of 10−4 g/L was used for promoting osteogenic differentiation of BMSCs in subsequent analyses. At this concentration, AR increased the levels of NO and cGMP, and such effects could be blocked by the estrogen receptor antagonist (ICI 182,780) and nitric oxide synthase inhibitor (L-NAME). Conclusion. AR induced osteogenic differentiation of rat BMSCs through the ER/NO/cGMP signaling pathway. This finding provides the theoretical foundation for the mechanism of AR in the treatment of postmenopausal osteoporosis.
doi:10.1155/2016/2615620
PMCID: PMC5126437  PMID: 27974901
2.  Automated glycan sequencing from tandem mass spectra of N-linked glycopeptides 
Analytical chemistry  2016;88(11):5725-5732.
Mass spectrometry has become a routine experimental tool for proteomic biomarker analysis of human blood samples, partly due to the large availability of informatics tools. As one of the most common protein post-translational modifications (PTMs) in mammals, protein glycosylation has been observed to alter in multiple human diseases, and thus may potentially be candidate markers of disease progression. While mass spectrometry instrumentation has seen advancements in capabilities, discovering glycosylation-related markers using existing software is currently not straightforward. Complete characterization of protein glycosylation requires the identification of intact glycopeptides in samples, including identification of the modification site as well as the structure of the attached glycans. In this paper, we present GlycoSeq, an open-source software tool that implements a heuristic iterated glycan sequencing algorithm coupled with prior knowledge for automated elucidation of the glycan structure within a glycopeptide from its collision-induced dissociation tandem mass spectrum. GlycoSeq employs rules of glycosidic linkage as defined by glycan synthetic pathways to eliminate improbable glycan structures and build reasonable glycan trees. We tested the tool on two sets of tandem mass spectra of N-linked glycopeptides cell lines acquired from breast cancer patients. After employing enzymatic specificity within the N-linked glycan synthetic pathway, the sequencing results of GlycoSeq were highly consistent with the manually curated glycan structures. Hence, GlycoSeq is ready to be used for the characterization of glycan structures in glycopeptides from MS/MS analysis. GlycoSeq is released as open source software at https://github.com/chpaul/GlycoSeq/.
Graphical Abstract
doi:10.1021/acs.analchem.5b04858
PMCID: PMC4899231  PMID: 27111718
glycan sequencing; N-linked glycopeptides; tandem mass spectrometry; computer software
3.  High‐Performance Polymer Solar Cells Based on a Wide‐Bandgap Polymer Containing Pyrrolo[3,4‐f]benzotriazole‐5,7‐dione with a Power Conversion Efficiency of 8.63% 
Advanced Science  2016;3(9):1600032.
A novel donor–acceptor type conjugated polymer based on a building block of 4,8‐di(thien‐2‐yl)‐6‐octyl‐2‐octyl‐5H‐pyrrolo[3,4‐f]benzotriazole‐5,7(6H)‐dione (TZBI) as the acceptor unit and 4,8‐bis(5‐(2‐ethylhexyl)thiophen‐2‐yl)­benzo­[1,2‐b:4,5‐b′]dithiophene as the donor unit, named as PTZBIBDT, is developed and used as an electron‐donating material in bulk‐heterojunction polymer solar cells. The resulting copolymer exhibits a wide bandgap of 1.81 eV along with relatively deep highest occupied molecular orbital energy level of −5.34 eV. Based on the optimized processing conditions, including thermal annealing, and the use of a water/alcohol cathode interlayer, the single‐junction polymer solar cell based on PTZBIBDT:PC71BM ([6,6]‐phenyl‐C71‐butyric acid methyl ester) blend film affords a power conversion efficiency of 8.63% with an open‐circuit voltage of 0.87 V, a short circuit current of 13.50 mA cm−2, and a fill factor of 73.95%, which is among the highest values reported for wide‐bandgap polymers‐based single‐junction organic solar cells. The morphology studies on the PTZBIBDT:PC71BM blend film indicate that a fibrillar network can be formed and the extent of phase separation can be mani­pulated by thermal annealing. These results indicate that the TZBI unit is a very promising building block for the synthesis of wide‐bandgap polymers for high‐performance single‐junction and tandem (or multijunction) organic solar cells.
doi:10.1002/advs.201600032
PMCID: PMC5039964  PMID: 27711267
polymer solar cells; pyrrolo[3,4‐f]benzotriazole‐5,7‐dione; wide‐bandgap polymer
4.  Polymer Solar Cells: High‐Performance Polymer Solar Cells Based on a Wide‐Bandgap Polymer Containing Pyrrolo[3,4‐f]benzotriazole‐5,7‐dione with a Power Conversion Efficiency of 8.63% (Adv. Sci. 9/2016) 
Advanced Science  2016;3(9):n/a.
In article 1600032, an efficient new wide‐bandgap polymer based on a novel moiety of pyrrolo[3,4‐f]benzotriazole‐5,7‐dione (TZBI) is developed by Lei Ying, Feng Lui, Thomas P. Russel, Fei Huang, and co‐workers. The new chemistry enables fine electronic structure tuning and solution‐processed single‐junction polymer solar cells provided a remarkable power conversion efficiency of 8.63%. Full electrical and structural characterization reveales that TZBI is a promising building block for the application in highly efficient organic photovoltaics.
doi:10.1002/advs.201670049
PMCID: PMC5039979
polymer solar cells; pyrrolo[3,4‐f]benzotriazole‐5,7‐dione; wide‐bandgap polymer
5.  Effects of Mechanical Stretch on Cell Proliferation and Matrix Formation of Mesenchymal Stem Cell and Anterior Cruciate Ligament Fibroblast 
Stem Cells International  2016;2016:9842075.
Mesenchymal stem cells (MSCs) and fibroblasts are two major seed cells for ligament tissue engineering. To understand the effects of mechanical stimulation on these cells and to develop effective approaches for cell therapy, it is necessary to investigate the biological effects of various mechanical loading conditions on cells. In this study, fibroblasts and MSCs were tested and compared under a novel Uniflex/Bioflex culture system that might mimic mechanical strain in ligament tissue. The cells were uniaxially or radially stretched with different strains (5%, 10%, and 15%) at 0.1, 0.5, and 1.0 Hz. The cell proliferation and collagen production were compared to find the optimal parameters. The results indicated that uniaxial stretch (15% at 0.5 Hz; 10% at 1.0 Hz) showed positive effects on fibroblast. The uniaxial strains (5%, 10%, and 15%) at 0.5 Hz and 10% strain at 1.0 Hz were favorable for MSCs. Radial strain did not have significant effect on fibroblast. On the contrary, the radial strains (5%, 10%, and 15%) at 0.1 Hz had positive effects on MSCs. This study suggested that fibroblasts and MSCs had their own appropriate mechanical stimulatory parameters. These specific parameters potentially provide fundamental knowledge for future cell-based ligament regeneration.
doi:10.1155/2016/9842075
PMCID: PMC4976179  PMID: 27525012
6.  Alteration of protein prenylation promotes spermatogonial differentiation and exhausts spermatogonial stem cells in newborn mice 
Scientific Reports  2016;6:28917.
Spermatogenesis in adulthood depends on the successful neonatal establishment of the spermatogonial stem cell (SSC) pool and gradual differentiation during puberty. The stage-dependent changes in protein prenylation in the seminiferous epithelium might be important during the first round of spermatogenesis before sexual maturation, but the mechanisms are unclear. We have previous found that altered prenylation in Sertoli cells induced spermatogonial apoptosis in the neonatal testis, resulting in adult infertility. Now we further explored the role of protein prenylation in germ cells, using a conditional deletion of geranylgeranyl diphosphate synthase (Ggpps) in embryonic stage and postmeiotic stage respectively. We observed infertility of Ggpps−/− Ddx4-Cre mice that displayed a Sertoli-cell-only syndrome phenotype, which resulted from abnormal spermatogonial differentiation and SSC depletion during the prepubertal stage. Analysis of morphological characteristics and cell-specific markers revealed that spermatogonial differentiation was enhanced from as early as the 7th postnatal day in the first round of spermatogenesis. Studies of the molecular mechanisms indicated that Ggpps deletion enhanced Rheb farnesylation, which subsequently activated mTORC1 and facilitated spermatogonial differentiation. In conclusion, the prenylation balance in germ cells is crucial for spermatogonial differentiation fate decision during the prepubertal stage, and the disruption of this process results in primary infertility.
doi:10.1038/srep28917
PMCID: PMC4931501  PMID: 27374985
7.  LC-MS/MS based Serum Proteomics for Identification of Candidate Biomarkers for Hepatocellular Carcinoma 
Proteomics  2015;15(13):2369-2381.
Associating changes in protein levels with the onset of cancer has been widely investigated to identify clinically relevant diagnostic biomarkers. In the present study, we analyzed sera from 205 patients recruited in the U.S. and Egypt for biomarker discovery using label-free proteomic analysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). We performed untargeted proteomic analysis of sera to identify candidate proteins with statistically significant differences between hepatocellular carcinoma (HCC) and patients with liver cirrhosis. We further evaluated the significance of 101 proteins in sera from the same 205 patients through targeted quantitation by multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer. This led to the identification of 21 candidate protein biomarkers that were significantly altered in both the U.S. and Egyptian cohorts. Among the 21 candidates, 10 were previously reported as HCC-associated proteins (eight exhibiting consistent trends with our observation), whereas 11 are new candidates discovered by this study. Pathway analysis based on the significant proteins reveals up-regulation of the complement and coagulation cascades pathway and down-regulation of the antigen processing and presentation pathway in HCC cases versus patients with liver cirrhosis. The results of this study demonstrate the power of combining untargeted and targeted quantitation methods for a comprehensive serum proteomic analysis, to evaluate changes in protein levels and discover novel diagnostic biomarkers.
doi:10.1002/pmic.201400364
PMCID: PMC4490019  PMID: 25778709
Cancer biomarker discovery; Hepatocellular carcinoma; Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS); Liver cirrhosis; Multiple reaction monitoring
8.  Segmentation of White Blood Cell from Acute Lymphoblastic Leukemia Images Using Dual-Threshold Method 
We propose a dual-threshold method based on a strategic combination of RGB and HSV color space for white blood cell (WBC) segmentation. The proposed method consists of three main parts: preprocessing, threshold segmentation, and postprocessing. In the preprocessing part, we get two images for further processing: one contrast-stretched gray image and one H component image from transformed HSV color space. In the threshold segmentation part, a dual-threshold method is proposed for improving the conventional single-threshold approaches and a golden section search method is used for determining the optimal thresholds. For the postprocessing part, mathematical morphology and median filtering are utilized to denoise and remove incomplete WBCs. The proposed method was tested in segmenting the lymphoblasts on a public Acute Lymphoblastic Leukemia (ALL) image dataset. The results show that the performance of the proposed method is better than single-threshold approach independently performed in RGB and HSV color space and the overall single WBC segmentation accuracy reaches 97.85%, showing a good prospect in subsequent lymphoblast classification and ALL diagnosis.
doi:10.1155/2016/9514707
PMCID: PMC4893444  PMID: 27313659
9.  High‐Performance Polymer Solar Cells Based on a Wide‐Bandgap Polymer Containing Pyrrolo[3,4‐f]benzotriazole‐5,7‐dione with a Power Conversion Efficiency of 8.63% 
Advanced Science  2016;3(9):1600032.
A novel donor–acceptor type conjugated polymer based on a building block of 4,8‐di(thien‐2‐yl)‐6‐octyl‐2‐octyl‐5H‐pyrrolo[3,4‐f]benzotriazole‐5,7(6H)‐dione (TZBI) as the acceptor unit and 4,8‐bis(5‐(2‐ethylhexyl)thiophen‐2‐yl)­benzo­[1,2‐b:4,5‐b′]dithiophene as the donor unit, named as PTZBIBDT, is developed and used as an electron‐donating material in bulk‐heterojunction polymer solar cells. The resulting copolymer exhibits a wide bandgap of 1.81 eV along with relatively deep highest occupied molecular orbital energy level of −5.34 eV. Based on the optimized processing conditions, including thermal annealing, and the use of a water/alcohol cathode interlayer, the single‐junction polymer solar cell based on PTZBIBDT:PC71BM ([6,6]‐phenyl‐C71‐butyric acid methyl ester) blend film affords a power conversion efficiency of 8.63% with an open‐circuit voltage of 0.87 V, a short circuit current of 13.50 mA cm−2, and a fill factor of 73.95%, which is among the highest values reported for wide‐bandgap polymers‐based single‐junction organic solar cells. The morphology studies on the PTZBIBDT:PC71BM blend film indicate that a fibrillar network can be formed and the extent of phase separation can be mani­pulated by thermal annealing. These results indicate that the TZBI unit is a very promising building block for the synthesis of wide‐bandgap polymers for high‐performance single‐junction and tandem (or multijunction) organic solar cells.
doi:10.1002/advs.201600032
PMCID: PMC5039964  PMID: 27711267
polymer solar cells; pyrrolo[3,4‐f]benzotriazole‐5,7‐dione; wide‐bandgap polymer
10.  The Effect of Chronic Methamphetamine Exposure on the Hippocampal and Olfactory Bulb Neuroproteomes of Rats 
PLoS ONE  2016;11(4):e0151034.
Nowadays, drug abuse and addiction are serious public health problems in the USA. Methamphetamine (METH) is one of the most abused drugs and is known to cause brain damage after repeated exposure. In this paper, we conducted a neuroproteomic study to evaluate METH-induced brain protein dynamics, following a two-week chronic regimen of an escalating dose of METH exposure. Proteins were extracted from rat brain hippocampal and olfactory bulb tissues and subjected to liquid chromatography-mass spectrometry (LC-MS/MS) analysis. Both shotgun and targeted proteomic analysis were performed. Protein quantification was initially based on comparing the spectral counts between METH exposed animals and their control counterparts. Quantitative differences were further confirmed through multiple reaction monitoring (MRM) LC-MS/MS experiments. According to the quantitative results, the expression of 18 proteins (11 in the hippocampus and 7 in the olfactory bulb) underwent a significant alteration as a result of exposing rats to METH. 13 of these proteins were up-regulated after METH exposure while 5 were down-regulated. The altered proteins belonging to different structural and functional families were involved in processes such as cell death, inflammation, oxidation, and apoptosis.
doi:10.1371/journal.pone.0151034
PMCID: PMC4833297  PMID: 27082425
11.  A randomized trial of the efficacy and safety of quilizumab in adults with inadequately controlled allergic asthma 
Respiratory Research  2016;17:29.
Background
Quilizumab, a humanized IgG1 monoclonal antibody, targets the M1-prime segment of membrane-expressed IgE, leading to depletion of IgE-switched and memory B cells. In patients with mild asthma, quilizumab reduced serum IgE and attenuated the early and late asthmatic reaction following whole lung allergen challenge. This study evaluated the efficacy and safety of quilizumab in adults with allergic asthma, inadequately controlled despite high-dose inhaled corticosteroids (ICS) and a second controller.
Methods
Five hundred seventy-eight patients were randomized to monthly or quarterly dosing regimens of subcutaneous quilizumab or placebo for 36 weeks, with a 48-week safety follow-up. Quilizumab was evaluated for effects on the rate of asthma exacerbations, lung function, patient symptoms, serum IgE, and pharmacokinetics. Exploratory analyses were conducted on biomarker subgroups (periostin, blood eosinophils, serum IgE, and exhaled nitric oxide).
Results
Quilizumab was well tolerated and reduced serum total and allergen-specific IgE by 30–40 %, but had no impact on asthma exacerbations, lung function, or patient-reported symptom measures. At Week 36, the 300 mg monthly quilizumab group showed a 19.6 % reduction (p = 0.38) in the asthma exacerbation rate relative to placebo, but this was neither statistically nor clinically significant. Biomarker subgroups did not reveal meaningful efficacy benefits following quilizumab treatment.
Conclusions
Quilizumab had an acceptable safety profile and reduced serum IgE. However, targeting the IgE pathway via depletion of IgE-switched and memory B cells was not sufficient for a clinically meaningful benefit for adults with allergic asthma uncontrolled by standard therapy.
Trial registration
ClinicalTrials.gov NCT01582503
Electronic supplementary material
The online version of this article (doi:10.1186/s12931-016-0347-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s12931-016-0347-2
PMCID: PMC4797126  PMID: 26993628
Allergic asthma; Biomarkers; COSTA; IgE; M1 prime; Quilizumab; Exacerbations; FEV1
12.  Interaction between hexon and L4-100K determines virus rescue and growth of hexon-chimeric recombinant Ad5 vectors 
Scientific Reports  2016;6:22464.
The immunogenicity of recombinant adenovirus serotype 5 (rAd5) vectors has been shown to be suppressed by neutralizing antibodies (NAbs) directed primarily against hexon hypervariable regions (HVRs). Preexisting immunity can be circumvented by replacing HVRs of rAd5 hexon with those derived from alternate adenovirus serotypes. However, chimeric modification of rAd5 hexon HVRs tends to cause low packaging efficiency or low proliferation of rAd5 vectors, but the related mechanism remains unclear. In this study, several Ad5-based vectors with precise replacement of HVRs with those derived from Ad37 and Ad43 were generated. We first observed that a HVR-exchanged rAd5 vector displayed a higher efficacy of the recombinant virus rescue and growth improvement compared with the rAd5 vector, although most hexon-chimeric rAd5 vectors constructed by us and other groups have proven to be nonviable or growth defective. We therefore evaluated the structural stability of the chimeric hexons and their interactions with the L4-100K chaperone. We showed that the viability of hexon-chimeric Ad5 vectors was not attributed to the structural stability of the chimeric hexon, but rather to the hexon maturation which was assisted by L4-100K. Our results suggested that the intricate interaction between hexon and L4-100K would determine the virus rescue and proliferation efficiency of hexon-chimeric rAd5 vectors.
doi:10.1038/srep22464
PMCID: PMC4776158  PMID: 26934960
13.  Serological Evaluation of Immunity to the Varicella-Zoster Virus Based on a Novel Competitive Enzyme-Linked Immunosorbent Assay 
Scientific Reports  2016;6:20577.
Varicella-zoster virus (VZV) is a highly contagious agent of varicella and herpes zoster. Varicella can be lethal to immunocompromised patients, babies, HIV patients and other adults with impaired immunity. Serological evaluation of immunity to VZV will help determine which individuals are susceptible and evaluate vaccine effectiveness. A collection of 110 monoclonal antibodies (mAbs) were obtained by immunization of mice with membrane proteins or cell-free virus. The mAbs were well characterized, and a competitive sandwich ELISA (capture mAb: 8H6; labelling mAb: 1B11) was established to determine neutralizing antibodies in human serum with reference to the FAMA test. A total of 920 human sera were evaluated. The competitive sandwich ELISA showed a sensitivity of 95.6%, specificity of 99.77% and coincidence of 97.61% compared with the fluorescent-antibody-to-membrane-antigen (FAMA) test. The capture mAb 8H6 was characterized as a specific mAb for VZV ORF9, a membrane-associated tegument protein that interacts with glycoprotein E (gE), glycoprotein B (gB) and glycoprotein C (gC). The labelling mAb 1B11 was characterized as a complement-dependent neutralizing mAb specific for the immune-dominant epitope located on gE, not on other VZV glycoproteins. The established competitive sandwich ELISA could be used as a rapid and high-throughput method for evaluating immunity to VZV.
doi:10.1038/srep20577
PMCID: PMC4744930  PMID: 26853741
14.  World checklist of hornworts and liverworts 
PhytoKeys  2016;1-828.
A working checklist of accepted taxa worldwide is vital in achieving the goal of developing an online flora of all known plants by 2020 as part of the Global Strategy for Plant Conservation. We here present the first-ever worldwide checklist for liverworts (Marchantiophyta) and hornworts (Anthocerotophyta) that includes 7486 species in 398 genera representing 92 families from the two phyla. The checklist has far reaching implications and applications, including providing a valuable tool for taxonomists and systematists, analyzing phytogeographic and diversity patterns, aiding in the assessment of floristic and taxonomic knowledge, and identifying geographical gaps in our understanding of the global liverwort and hornwort flora. The checklist is derived from a working data set centralizing nomenclature, taxonomy and geography on a global scale. Prior to this effort a lack of centralization has been a major impediment for the study and analysis of species richness, conservation and systematic research at both regional and global scales. The success of this checklist, initiated in 2008, has been underpinned by its community approach involving taxonomic specialists working towards a consensus on taxonomy, nomenclature and distribution.
doi:10.3897/phytokeys.59.6261
PMCID: PMC4758082  PMID: 26929706
Marchantiophyta; Anthocerophyta; nomenclature; taxonomy
15.  Distinct mechanisms of cell-kill by triapine and its terminally dimethylated derivative Dp44mT due to a loss or gain of activity of their copper(II) complexes 
Biochemical pharmacology  2014;91(3):312-322.
Triapine, currently being evaluated as an antitumor agent in phase II clinical trials, and its terminally dimethylated derivative Dp44mT share the α-pyridyl thiosemicarbazone backbone that functions as ligands for transition metal ions. Yet, Dp44mT is approximately 100-fold more potent than triapine in cytotoxicity assays. The aims of this study were to elucidate the mechanisms underlying their potency disparity and to determine their kinetics of cell-kill in culture to aid in the formulation of their clinical dosing schedules. The addition of Cu2+ inactivated triapine in a 1:1 stoichiometric fashion, while it potentiated the cytotoxicity of Dp44mT. Clonogenic assays after finite-time drug-exposure revealed that triapine produced cell-kill in two phases, one completed within 20 min that caused limited cell-kill, and the other occurring after 16 h of exposure that produced extensive cell-kill. The ribonucleotide reductase inhibitor triapine at 0.4 µM caused immediate complete arrest of DNA synthesis, whereas Dp44mT at this concentration did not appreciably inhibit DNA synthesis. The inhibition of DNA synthesis by triapine was reversible upon its removal from the medium. Cell death after 16 h exposure to triapine paralleled the appearance of phospho-(γ)H2AX, a marker of DNA double-strand breaks induced by collapse of DNA replication forks after prolonged replication arrest. In contrast to triapine, Dp44mT produced robust cell-kill within 1 h in a concentration-dependent manner. The short-term action of both agents was prevented by thiols, indicative of the involvement of reactive oxygen species. The time dependency in the production of cell-kill by triapine should be considered in treatment regimens.
doi:10.1016/j.bcp.2014.08.006
PMCID: PMC4163625  PMID: 25130544
Triapine (3-AP, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone); Dp44mT (di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone); Metal coordination; DNA replication stress; DNA double-strand breaks; Reactive oxygen species
16.  Natural attenuation model and biodegradation for 1,1,1-trichloroethane contaminant in shallow groundwater 
Natural attenuation is an effective and feasible technology for controlling groundwater contamination. This study investigated the potential effectiveness and mechanisms of natural attenuation of 1,1,1-trichloroethane (TCA) contaminants in shallow groundwater in Shanghai by using a column simulation experiment, reactive transport model, and 16S rRNA gene clone library. The results indicated that the majority of the contaminant mass was present at 2–6 m in depth, the contaminated area was approximately 1000 m × 1000 m, and natural attenuation processes were occurring at the site. The effluent breakthrough curves from the column experiments demonstrated that the effectiveness of TCA natural attenuation in the groundwater accorded with the advection-dispersion-reaction equation. The kinetic parameter of adsorption and biotic dehydrochlorination of TCA was 0.068 m3/kg and 0.0045 d–1. The contamination plume was predicted to diminish and the maximum concentration of TCA decreased to 280 μg/L. The bacterial community during TCA degradation in groundwater belonged to Trichococcus, Geobacteraceae, Geobacter, Mucilaginibacter, and Arthrobacter.
doi:10.3389/fmicb.2015.00839
PMCID: PMC4548683  PMID: 26379629
1,1,1-trichloroethane; contaminated groundwater; natural attenuation; bacterial communities; dechlorination
18.  LC–MS/MS Quantitation of Esophagus Disease Blood Serum Glycoproteins by Enrichment with Hydrazide Chemistry and Lectin Affinity Chromatography 
Journal of Proteome Research  2014;13(11):4808-4820.
Changes in glycosylation have been shown to have a profound correlation with development/malignancy in many cancer types. Currently, two major enrichment techniques have been widely applied in glycoproteomics, namely, lectin affinity chromatography (LAC)-based and hydrazide chemistry (HC)-based enrichments. Here we report the LC–MS/MS quantitative analyses of human blood serum glycoproteins and glycopeptides associated with esophageal diseases by LAC- and HC-based enrichment. The separate and complementary qualitative and quantitative data analyses of protein glycosylation were performed using both enrichment techniques. Chemometric and statistical evaluations, PCA plots, or ANOVA test, respectively, were employed to determine and confirm candidate cancer-associated glycoprotein/glycopeptide biomarkers. Out of 139, 59 common glycoproteins (42% overlap) were observed in both enrichment techniques. This overlap is very similar to previously published studies. The quantitation and evaluation of significantly changed glycoproteins/glycopeptides are complementary between LAC and HC enrichments. LC–ESI–MS/MS analyses indicated that 7 glycoproteins enriched by LAC and 11 glycoproteins enriched by HC showed significantly different abundances between disease-free and disease cohorts. Multiple reaction monitoring quantitation resulted in 13 glycopeptides by LAC enrichment and 10 glycosylation sites by HC enrichment to be statistically different among disease cohorts.
doi:10.1021/pr500570m
PMCID: PMC4227547  PMID: 25134008
19.  Effect of Qing’e formula on the in vitro differentiation of bone marrow-derived mesenchymal stem cells from proximal femurs of postmenopausal osteoporotic mice 
Background
Qing’e formula (QEF), prepared from an ancient Chinese recipe, was previously suggested to regulate bone metabolism and improve bone mineral density in patients with osteoporosis. To study the effects of medicated serum containing QEF on the in vitro differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) isolated from the proximal femurs of postmenopausal osteoporosis (PMOP) mice.
Methods
Using an established mouse model of PMOP, mononuclear cells were isolated from the bone marrow present in the proximal femurs and cultured. PMOP mice were also randomly divided into four groups: the untreated group (Group A) and the groups treated with respectively low (Group B), medium (Group C), and high (Group D) concentrations of QEF. Serum was isolated from each and used to treat the cultured BMSCs in conjunction with recombinant human bone morphogenetic protein-2 (rhBMP-2). Cell morphology, proliferation rates, intracellular alkaline phosphatase (ALP) activity, and transforming growth factor-beta 1 (TGF-β1) mRNA expression were evaluated.
Results
QEF-treated serum, particularly that containing moderate and high concentrations, appears to enhance the rhBMP-2-mediated changes in cell morphology, proliferation, and differentiation (determined via the expression of TGF-β1 mRNA and ALP activity) observed in the BMSCs isolated from PMOP mice.
Conclusions
QEF may play a role in the prevention and treatment of PMOP by enhancing the activity of rhBMP-2.
doi:10.1186/s12906-015-0777-2
PMCID: PMC4513391  PMID: 26205885
Osteoporosis; Bone marrow-derived mesenchymal stem cells; Qing’e formula; Transforming growth factor β
20.  Influence of Glutathione and Glutathione S-transferases on DNA Interstrand Cross-Link Formation by 1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine, the Active Anticancer Moiety Generated by Laromustine 
Chemical Research in Toxicology  2014;27(8):1440-1449.
Prodrugs of 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE) are promising anticancer agents. The 90CE moiety is a readily latentiated, short-lived (t1/2 ∼ 30 s) chloroethylating agent that can generate high yields of oxophilic electrophiles responsible for the chloroethylation of the O-6 position of guanine in DNA. These guanine O-6 alkylations are believed to be responsible for the therapeutic effects of 90CE and its prodrugs. Thus, 90CE demonstrates high selectivity toward tumors with diminished levels of O6-alkylguanine-DNA alkyltransferase (MGMT), the resistance protein responsible for O6-alkylguanine repair. The formation of O6-(2-chloroethyl)guanine lesions ultimately leads to the generation of highly cytotoxic 1-(N3-cytosinyl),-2-(N1-guaninyl)ethane DNA interstrand cross-links via N1,O6-ethanoguanine intermediates. The anticancer activity arising from this sequence of reactions is thus identical to this component of the anticancer activity of the clinically used chloroethylnitrosoureas. Herein, we evaluate the ability of glutathione (GSH) and other low molecular weight thiols, as well as GSH coupled with various glutathione S-transferase enzymes (GSTs) to attenuate the final yields of cross-links generated by 90CE when added prior to or immediately following the initial chloroethylation step to determine the major point(s) of interaction. In contrast to studies utilizing BCNU as a chloroethylating agent by others, GSH (or GSH/GST) did not appreciably quench DNA interstrand cross-link precursors. While thiols alone offered little protection at either alkylation step, the GSH/GST couple was able to diminish the initial yields of cross-link precursors. 90CE exhibited a very different GST isoenzyme susceptibility to that reported for BCNU, this could have important implications in the relative resistance of tumor cells to these agents. The protection afforded by GSH/GST was compared to that produced by MGMT.
doi:10.1021/tx500197t
PMCID: PMC4137992  PMID: 25012050
21.  Assessment of the Impact of Zoledronic Acid on Ovariectomized Osteoporosis Model Using Micro-CT Scanning 
PLoS ONE  2015;10(7):e0132104.
Purpose/Objective
Prompted by preliminary findings, this study was conducted to investigate the impact of zoledronic acid on the cancellous bone microstructure and its effect on the level of β-catenin in a mouse model of postmenopausal osteoporosis.
Methods and Materials
96 8-week-old specific-pathogen-free C57BL/6 mice were randomly divided into 4 groups (24 per group): a sham group, an ovariectomized osteoporosis model group, an estradiol-treated group, and a zoledronic acid-treated group. Five months after surgery, the third lumbar vertebra and left femur of the animals were dissected and scanned using micro-computed tomography (micro-CT) to acquire three-dimensional imagery of their cancellous bone microstructure. The impact of ovariectomy, the effect of estradiol, and the effect of zoledronic acid intervention on cancellous bone microstructure, as well as on the expression of β-catenin, were evaluated.
Results
The estradiol-treated and the zoledronic acid-treated group exhibited a significant increase in the bone volume fraction, trabecular number, trabecular thickness, bone surface to bone volume ratio (BS/BV), and β-catenin expression, when compared with those of the control group (P <0.01). In contrast, the structure model index, trabecular separation, and BS/BV were significantly lower compared with those of the model group (P <0.01). No differences were observed in the above parameters between animals of the zoledronic acid-treated and the estradiol-treated group.
Conclusion
These results suggest that increased β-catenin expression may be the mechanism underlying zoledronic acid-related improvement in the cancellous bone microstructure in ovariectomized mice. Our findings provide a scientific rationale for using zoledronic acid as a therapeutic intervention to prevent bone loss in post-menopausal women.
doi:10.1371/journal.pone.0132104
PMCID: PMC4492783  PMID: 26148020
22.  Characterization of the Kallikrein-Kinin System Post Chemical Neuronal Injury: An In Vitro Biochemical and Neuroproteomics Assessment 
PLoS ONE  2015;10(6):e0128601.
Traumatic Brain Injury (TBI) is the result of a mechanical impact on the brain provoking mild, moderate or severe symptoms. It is acknowledged that TBI leads to apoptotic and necrotic cell death; however, the exact mechanism by which brain trauma leads to neural injury is not fully elucidated. Some studies have highlighted the pivotal role of the Kallikrein-Kinin System (KKS) in brain trauma but the results are still controversial and inconclusive. In this study, we investigated both the expression and the role of Bradykinin 1 and 2 receptors (B1R and B2R), in mediating neuronal injury under chemical neurotoxicity paradigm in PC12 cell lines. The neuronal cell line PC12 was treated with the apoptotic drug Staurosporine (STS) to induce cell death. Intracellular calcium release was evaluated by Fluo 4-AM staining and showed that inhibition of the B2R prevented calcium release following STS treatment. Differential analyses utilizing immunofluorescence, Western blot and Real-time Polymerase Chain Reaction revealed an upregulation of both bradykinin receptors occurring at 3h and 12h post-STS treatment, but with a higher induction of B2R compared to B1R. This implies that STS-mediated apoptosis in PC12 cells is mainly conducted through B2R and partly via B1R. Finally, a neuroproteomics approach was conducted to find relevant proteins associated to STS and KKS in PC12 cells. Neuroproteomics results confirmed the presence of an inflammatory response leading to cell death during apoptosis-mediated STS treatment; however, a “survival” capacity was shown following inhibition of B2R coupled with STS treatment. Our data suggest that B2R is a key player in the inflammatory pathway following STS-mediated apoptosis in PC12 cells and its inhibition may represent a potential therapeutic tool in TBI.
doi:10.1371/journal.pone.0128601
PMCID: PMC4457722  PMID: 26047500
23.  Biomarker-guided sequential targeted therapies to overcome therapy resistance in rapidly evolving highly aggressive mammary tumors 
Cell Research  2014;24(5):542-559.
Combinatorial targeted therapies are more effective in treating cancer by blocking by-pass mechanisms or inducing synthetic lethality. However, their clinical application is hampered by resistance and toxicity. To meet this important challenge, we developed and tested a novel concept of biomarker-guided sequential applications of various targeted therapies using ErbB2-overexpressing/PTEN-low, highly aggressive breast cancer as our model. Strikingly, sustained activation of ErbB2 and downstream pathways drives trastuzumab resistance in both PTEN-low/trastuzumab-resistant breast cancers from patients and mammary tumors with intratumoral heterogeneity from genetically-engineered mice. Although lapatinib initially inhibited trastuzumab-resistant mouse tumors, tumors by-passed the inhibition by activating the PI3K/mTOR signaling network as shown by the quantitative protein arrays. Interestingly, activation of the mTOR pathway was also observed in neoadjuvant lapatinib-treated patients manifesting lapatinib resistance. Trastuzumab + lapatinib resistance was effectively overcome by sequential application of a PI3K/mTOR dual kinase inhibitor (BEZ235) with no significant toxicity. However, our p-RTK array analysis demonstrated that BEZ235 treatment led to increased ErbB2 expression and phosphorylation in genetically-engineered mouse tumors and in 3-D, but not 2-D, culture, leading to BEZ235 resistance. Mechanistically, we identified ErbB2 protein stabilization and activation as a novel mechanism of BEZ235 resistance, which was reversed by subsequent treatment with lapatinib + BEZ235 combination. Remarkably, this sequential application of targeted therapies guided by biomarker changes in the tumors rapidly evolving resistance doubled the life-span of mice bearing exceedingly aggressive tumors. This fundamentally novel approach of using targeted therapies in a sequential order can effectively target and reprogram the signaling networks in cancers evolving resistance during treatment.
doi:10.1038/cr.2014.37
PMCID: PMC4011340  PMID: 24675532
sequential therapy; tumor evolution; targeted therapy; trastuzumab resistance; BEZ235; ErbB2 stabilization
24.  Influence of Phosphate and Phosphoesters on the Decomposition Pathway of 1,2-Bis(methylsulfonyl)-1-(2-chloroethyhydrazine (90CE), the Active Anticancer Moiety Generated by Laromustine, KS119, and KS119W 
Chemical Research in Toxicology  2014;27(5):818-833.
Prodrugs of the short-lived chloroethylating agent 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE) and its methylating analogue 1,2-bis(methylsulfonyl)-1-(methyl)hydrazine (KS90) are potentially useful anticancer agents. This class of agents frequently yields higher ratios of therapeutically active oxophilic electrophiles responsible for DNA O6-guanine alkylations to other electrophiles with lower therapeutic relevance than the nitrosoureas. This results in improved selectivity toward tumors with diminished levels of O6-alkylguanine-DNA alkyltransferase (MGMT), the resistance protein responsible for O6-alkylguanine repair. The formation of O6-(2-chloroethyl)guanine, which leads to the formation of a DNA–DNA interstrand cross-link, accounts for the bulk of the anticancer activity of 90CE prodrugs. Herein, we describe a new decomposition pathway that is available to 90CE but not to its methylating counterpart. This pathway appears to be subject to general/acid base catalysis with phosphate (Pi), phosphomonoesters, and phosphodiesters, being particularly effective. This pathway does not yield a chloroethylating species and results in a major change in nucleophile preference since thiophilic rather than oxophilic electrophiles are produced. Thus, a Pi concentration dependent decrease in DNA–DNA interstand cross-link formation was observed. Changes in 90CE decomposition products but not alkylation kinetics occurred in the presence of Pi since the prebranch point elimination of the N-1 methanesulfinate moiety remained the rate-limiting step. The Pi catalyzed route is expected to dominate at Pi and phosphoester concentrations totaling >25–35 mM. In view of the abundance of Pi and phosphoesters in cells, this pathway may have important effects on agent toxicity, tumor selectivity, and resistance to prodrugs of 90CE. Furthermore, it may be possible to design analogues that diminish this thiophile-generating pathway, which is likely superfluous at best and potentially detrimental to the targeting of hypoxic regions where Pi concentrations can be significantly elevated.
doi:10.1021/tx500004y
PMCID: PMC4033638  PMID: 24618018
25.  Tim-3 protects decidual stromal cells from toll-like receptor-mediated apoptosis and inflammatory reactions and promotes Th2 bias at the maternal-fetal interface 
Scientific Reports  2015;5:9013.
Toll-like receptors (TLRs) are important in mediating immune responses against various pathogens during pregnancy. However, uncontrolled TLR-triggered inflammation will endanger normal pregnancy, resulting in pregnancy loss. Therefore, maintenance of a moderate inflammatory response is crucial for successful pregnancy under conditions of infection. Here, we demonstrated significantly lowered expression of T-cell immunoglobulin and mucin domain 3 (Tim-3) in miscarried decidual stromal cells (DSCs), indicating that Tim-3 might play important roles in maintaining successful pregnancies. Activation of TLR signaling induced pro-inflammatory cytokine production and apoptosis of DSCs, which was accompanied by up-regulated Tim-3 expression. Tim-3, in turn, protected DSCs from TLR-mediated apoptosis in an ERK1/2 pathway-dependent manner. In addition, Tim-3 inhibited TLR signaling-induced inflammatory cytokine production by DSCs through suppressing NF-κB activation. Tim-3 increased production of T helper 2 (Th2)-type cytokines by DSCs and reversed the inhibitory effect of LPS on Th2 cytokine generation by up-regulation of interferon regulatory factor 4 expression. Tim-3 blockade abolished the effect of Tim-3 on the inflammatory response to LPS stimulation. Thus, Tim-3 signaling could represent a “self-control” mechanism in TLR-triggered inflammation during pregnancy. These findings identify Tim-3 as a key regulator of DSCs and suggest its potential as a target for the treatment of spontaneous abortion.
doi:10.1038/srep09013
PMCID: PMC4355741  PMID: 25757669

Results 1-25 (74)