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1.  Expression of Cystatin SN significantly correlates with recurrence, metastasis, and survival duration in surgically resected non-small cell lung cancer patients 
Scientific Reports  2015;5:8230.
Cystatin SN has been considered to be involved in human cancer, but its clinical significance in non-small cell lung cancer (NSCLC) has not been elucidated. The aim of this study was to evaluate the clinical value of Cystatin SN expression in patients with surgically resected NSCLCs. A retrospective analysis of 174 patients with surgically resected NSCLCs from April 2002 to March 2005 was performed with immunohistochemistry and fluorescence in situ hybridization to analyze the protein expression and amplification of Cystatin SN. The associations between Cystatin SN expression and recurrence, metastasis, and survival were investigated. In recurrence and metastasis analysis, compared with low-Cystatin SN expression NSCLCs, high expression tumors were more likely to recur and metastasize (P < 0.001). Disease-free survival (DFS) and overall survival (OS) were significantly prolonged in the low-Cystatin SN expression subgroup compared with the high-Cystatin SN expression subgroup (DFS, P < 0.001; OS, P = 0.001). A multivariate analysis confirmed that high expression of Cystatin SN was associated with poor survival (DFS, P = 0.001; OS, P = 0.006) and was an independent prognostic indicator. The present study indicates that high expression of Cystatin SN is a significant prognostic indicator of a higher rate of recurrence, metastatic risk, and poor survival in patients with surgically resected NSCLCs.
doi:10.1038/srep08230
PMCID: PMC4316172  PMID: 25648368
2.  Elucidation of the RamA Regulon in Klebsiella pneumoniae Reveals a Role in LPS Regulation 
PLoS Pathogens  2015;11(1):e1004627.
Klebsiella pneumoniae is a significant human pathogen, in part due to high rates of multidrug resistance. RamA is an intrinsic regulator in K. pneumoniae established to be important for the bacterial response to antimicrobial challenge; however, little is known about its possible wider regulatory role in this organism during infection. In this work, we demonstrate that RamA is a global transcriptional regulator that significantly perturbs the transcriptional landscape of K. pneumoniae, resulting in altered microbe-drug or microbe-host response. This is largely due to the direct regulation of 68 genes associated with a myriad of cellular functions. Importantly, RamA directly binds and activates the lpxC, lpxL-2 and lpxO genes associated with lipid A biosynthesis, thus resulting in modifications within the lipid A moiety of the lipopolysaccharide. RamA-mediated alterations decrease susceptibility to colistin E, polymyxin B and human cationic antimicrobial peptide LL-37. Increased RamA levels reduce K. pneumoniae adhesion and uptake into macrophages, which is supported by in vivo infection studies, that demonstrate increased systemic dissemination of ramA overexpressing K. pneumoniae. These data establish that RamA-mediated regulation directly perturbs microbial surface properties, including lipid A biosynthesis, which facilitate evasion from the innate host response. This highlights RamA as a global regulator that confers pathoadaptive phenotypes with implications for our understanding of the pathogenesis of Enterobacter, Salmonella and Citrobacter spp. that express orthologous RamA proteins.
Author Summary
Bacteria can rapidly evolve under antibiotic pressure to develop resistance, which occurs when target genes mutate, or when resistance-encoding genes are transferred. Alternatively, microbes can simply alter the levels of intrinsic proteins that allow the organism to “buy” time to resist antibiotic pressure. Klebsiella pneumoniae is a pathogen that causes significant blood stream or respiratory infections, but more importantly is a bacterium that is increasingly being reported as multidrug resistant. Our data demonstrate that RamA can trigger changes on the bacterial surface that allow Klebsiella to survive both antibiotic challenge, degradation by host immune peptides and resist phagocytosis. We demonstrate that the molecular basis of increased survival of ramA overexpressing K. pneumoniae, against host-derived factors is associated with RamA-driven alterations of the lipid A moiety of Klebsiella LPS. This modification is likely to be linked to Klebsiella’s ability to resist the host response so that it remains undetected by the immune system. The relevance of our work extends beyond RamA in Klebsiella as other pathogens such as Enterobacter spp and Salmonella spp. also produce this protein. Thus our overarching conclusion is that the intrinsic regulator, RamA perturbs host-microbe and microbe-drug interactions.
doi:10.1371/journal.ppat.1004627
PMCID: PMC4310594  PMID: 25633080
3.  Culture medium of bone marrow-derived human mesenchymal stem cells effects lymphatic endothelial cells and tumor lymph vessel formation 
Oncology Letters  2015;9(3):1221-1226.
Human bone marrow mesenchymal stem cells (hBM-MSCs) favor tumor growth and metastasis in vivo and in vitro. Neovascularization is involved in several pathological conditions, including tumor growth and metastasis. Previous studies have demonstrated that human bone marrow MSC-derived conditioned medium (hBM-MSC-CM) can promote tumor growth by inducing the expression of vascular epidermal growth factor (VEGF) in tumor cells. However, the effect of BM-MSCs on tumor lymph vessel formation has yet to be elucidated. In the present study, the effect of BM-MSCs on processes involved in lymph vessel formation, including tube formation, migration and proliferation, was investigated in human-derived lymphatic endothelial cells (HDLECs). It was identified that hBM-MSC-CM promoted the tube formation and migration of HDLECs. In addition, tumor cells were revealed to participate in lymph vessel formation. In the present study, the SGC-7901, HGC-27 and GFP-MCF-7 cell lines were treated with hBM-MSC-CM. The results demonstrated that the expression of the lymph-associated markers, prospero homeobox protein 1 and VEGF receptor-3, were increased in the SGC-7901 and HGC-27 cell lines, but not in the GFP-MCF-7 cells. The tube formation assay demonstrated that the HGC-27 cells treated with hBM-MSC-CM for 20 days underwent tube formation. These findings indicate that hBM-MSC-CM can promote tube formation in HDLECs and HGC-27 cells, which may be associated with lymph vessel formation during tumor growth and metastasis.
doi:10.3892/ol.2015.2868
PMCID: PMC4315037  PMID: 25663886
mesenchymal stem cell; lymph vessel; tumor growth
4.  Variations among Primary Care Physicians in Exercise Advice, Imaging, and Analgesics for Musculoskeletal Pain: Results from a Factorial Experiment 
Arthritis care & research  2014;66(1):147-156.
Objective
To examine whether medical decisions regarding evaluation and management of musculoskeletal pain conditions varied systematically by characteristics of the patient or provider.
Methods
We conducted a balanced factorial experiment among primary care physicians in the U.S. Physicians (N=192) viewed two videos of different patients (actors) presenting with pain: (1) undiagnosed sciatica symptoms or (2) diagnosed knee osteoarthritis. Systematic variations in patient gender, socioeconomic status (SES), race, physician gender and experience (<20 vs. ≥20 years in practice) permitted estimation of unconfounded effects. Analysis of variance was used to evaluate associations between patient or provider attributes and clinical decisions. Quality of decisions was defined based on the current recommendations of the ACR, American Pain Society, and clinical expert consensus.
Results
Despite current recommendations, under one-third of physicians would provide exercise advice (30.2% for osteoarthritis, 32.8% for sciatica). Physicians with fewer years in practice were more likely to provide advice on lifestyle changes, particularly exercise (P<0.01), and to prescribe NSAIDs for pain relief, both of which were appropriate and consistent with current recommendations for care. Newer physicians ordered fewer tests, particularly basic laboratory investigations or urinalysis. Test ordering decreased as organizational emphasis on business or profits increased. Patient factors and physician gender had no consistent effects on pain evaluation or treatment.
Conclusion
Physician education on disease management recommendations regarding exercise and analgesics, and implementation of quality measures may be useful, particularly for physicians with more years in practice.
doi:10.1002/acr.22143
PMCID: PMC4067704  PMID: 24376249
5.  Computational Approaches to Analyze and Predict Small Molecule Transport and Distribution at Cellular and Subcellular Levels 
Quantitative structure-activity relationship (QSAR) studies and mechanistic mathematical modeling approaches have been independently employed for analyzing and predicting the transport and distribution of small molecule chemical agents in living organisms. Both of these computational approaches have been useful to interpret experiments measuring the transport properties of small molecule chemical agents, in vitro and in vivo. Nevertheless, mechanistic cell-based pharmacokinetic models have been especially useful to guide the design of experiments probing the molecular pathways underlying small molecule transport phenomena. Unlike QSAR models, mechanistic models can be integrated from microscopic to macroscopic levels, to analyze the spatiotemporal dynamics of small molecule chemical agents from intracellular organelles to whole organs, well beyond the experiments and training data sets upon which the models are based. Based on differential equations, mechanistic models can also be integrated with other differential equations-based systems biology models of biochemical networks or signaling pathways. Although the origin and evolution of mathematical modeling approaches aimed at predicting drug transport and distribution has occurred independently from systems biology, we propose that the incorporation of mechanistic cell-based computational models of drug transport and distribution into a systems biology modeling framework is a logical next-step for the advancement of systems pharmacology research.
doi:10.1002/bdd.1879
PMCID: PMC3947293  PMID: 24218242
Cellular pharmacokinetics; Computational modeling; Drug Transport; Systems pharmacology
6.  Parthenolide Inhibits Cancer Stem-Like Side Population of Nasopharyngeal Carcinoma Cells via Suppression of the NF-κB/COX-2 Pathway 
Theranostics  2015;5(3):302-321.
Cancer stem cells play a central role in the pathogenesis of nasopharyngeal carcinoma and contribute to both disease initiation and relapse. In this study, cyclooxygenase-2 (COX-2) was found to regulate cancer stem-like side population cells of nasopharyngeal carcinoma cells and enhance cancer stem-like cells' characteristics such as higher colony formation efficiency and overexpression of stemness-associated genes. The regulatory effect of COX-2 on cancer stem-like characteristics may be mediated by ABCG2. COX-2 overexpression by a gain-of-function experiment increased the proportion of side population cells and their cancer stemness properties. The present study also demonstrated that in contrast to the classical chemotherapy drug 5-fluorouracil, which increased the proportion of side population cells and upregulated the expression of COX-2, parthenolide, a naturally occurring small molecule, preferentially targeted the side population cells of nasopharyngeal carcinoma cells and downregulated COX-2. Moreover, we found that the cancer stem-like cells' phenotype was suppressed by using COX-2 inhibitors NS-398 and CAY10404 or knocking down COX-2 with siRNA and shRNA. These findings suggest that COX-2 inhibition is the mechanism by which parthenolide induces cell death in the cancer stem-like cells of nasopharyngeal carcinoma. In addition, parthenolide exhibited an inhibitory effect on nuclear factor-kappa B (NF-κB) nucler translocation by suppressing both the phosphorylation of IκB kinase complex and IκBα degradation. Taken together, these results suggest that parthenolide may exert its cancer stem cell-targeted chemotherapy through the NF-κB/COX-2 pathway.
doi:10.7150/thno.8387
PMCID: PMC4279193  PMID: 25553117
parthenolide; nasopharyngeal carcinoma; cyclooxygenase-2; nuclear factor-kappa B; side population cells.
7.  Radiobiological characteristics of cancer stem cells from esophageal cancer cell lines 
World Journal of Gastroenterology : WJG  2014;20(48):18296-18305.
AIM: To study the cancer stem cell population in esophageal cancer cell lines KYSE-150 and TE-1 and identify whether the resulting stem-like spheroid cells display cancer stem cells and radiation resistance characteristics.
METHODS: A serum-free medium (SFM) suspension was used to culture esophageal cancer stem cell lines and enrich the esophageal stem-like spheres. A reverse transcription polymerase chain reaction assay was used to detect stem cell gene expression in the spheroid cells. Radiosensitivity of stem-like spheres and parental cells were evaluated by clonogenic assays. Furthermore, different cells after different doses of irradiation were tested to evaluate the change in sphere formation, cell cycle and CD44+CD271+ expression of tumor stem-like spheroid cells using flow cytometry before and after irradiation.
RESULTS: The cells were observed to generate an increased number of spheres in SFM with increasing cell passage. Radiation increased the rate of generation of stem-like spheres in both types of cells. The average survival fraction (SF2) of the cultured KYSE-150 compared with TE-1 stem-like spheres after 2 Gy of radiation was 0.81 ± 0.03 vs 0.87 ± 0.01 (P < 0.05), while the average SF2 of KYSE-150 compared with TE-1 parental cells was 0.69 ± 0.04 vs 0.80 ± 0.03, P < 0.05. In the esophageal parental cells, irradiation dose-dependently induced G2 arrest. Stem-like esophageal spheres were resistant to irradiation-induced G2 arrest without significant changes in the percentage population of irradiated stem-like cells. Under irradiation at 0, 4, and 8 Gy, the CD44+CD271+ cell percentage for KYSE150 parental cells was 1.08% ± 0.03% vs 1.29% ± 0.07% vs 1.11% ± 0.09%, respectively; the CD44+CD271+ cell percentage for TE1 parental cells was 1.16% ± 0.11% vs 0.97% ± 0.08% vs 1.45% ± 0.35%, respectively. The differences were not statistically significant. Under irradiation at 0, 4, and 8 Gy, the CD44+CD271+ cell percentage for KYSE-150 stem-like spheres was 35.83% ± 1.23% vs 44.9% ± 1.67% vs 57.77% ± 1.88%, respectively; the CD44+CD271+ cell percentage for TE1 stem-like spheres was 16.07% ± 0.91% vs 22.67% ± 1.12%, 16.07% ± 0.91% vs 33.27% ± 1.07%, respectively. The 4 and 8 Gy irradiated KYSE-150 and TE-1 stem-like spheres were compared with the 0 Gy irradiated group, and the differences were statistically significant (P < 0.05).
CONCLUSION: The KYSE-150 and TE-1 stem-like spheres are more radioresistant than their parental cells which may suggest that cancer stem cells are related to radioresistance.
doi:10.3748/wjg.v20.i48.18296
PMCID: PMC4277966  PMID: 25561796
Esophageal neoplasms; Radiation resistance; Neoplastic stem cell; Cell spheres; Cell cycle
8.  Multiple species of wild tree peonies gave rise to the ‘king of flowers’, Paeonia suffruticosa Andrews 
The origin of cultivated tree peonies, known as the ‘king of flowers' in China for more than 1000 years, has attracted considerable interest, but remained unsolved. Here, we conducted phylogenetic analyses of explicitly sampled traditional cultivars of tree peonies and all wild species from the shrubby section Moutan of the genus Paeonia based on sequences of 14 fast-evolved chloroplast regions and 25 presumably single-copy nuclear markers identified from RNA-seq data. The phylogeny of the wild species inferred from the nuclear markers was fully resolved and largely congruent with morphology and classification. The incongruence between the nuclear and chloroplast trees suggested that there had been gene flow between the wild species. The comparison of nuclear and chloroplast phylogenies including cultivars showed that the cultivated tree peonies originated from homoploid hybridization among five wild species. Since the origin, thousands of cultivated varieties have spread worldwide, whereas four parental species are currently endangered or on the verge of extinction. The documentation of extensive homoploid hybridization involved in tree peony domestication provides new insights into the mechanisms underlying the origins of garden ornamentals and the way of preserving natural genetic resources through domestication.
doi:10.1098/rspb.2014.1687
PMCID: PMC4240985  PMID: 25377453
conservation; hybridization; Paeonia; RNA-seq; species tree
9.  Meta-analysis of the efficacy of probiotics in Helicobacter pylori eradication therapy 
World Journal of Gastroenterology : WJG  2014;20(47):18013-18021.
AIM: To evaluate the role of probiotics in the standard triple Helicobacter pylori therapy.
METHODS: In this meta-analysis, we investigated the efficacy of probiotics in a standard triple H. pylori therapy in adults. Searches were mainly conducted in MEDLINE/PubMed, EMBASE, and the Cochrane Central Register of Controlled Trials. Fourteen studies met our criteria, and the quality of these studies was assessed using the Jadad scale. We used STATA version 12.0 to extract data and to calculate the odds ratios (ORs), which are presented with the corresponding 95% confidence intervals (CIs). The data are presented as forest plots.
RESULTS: The pooled ORs for the eradication rates calculated by intention-to-treat analysis and per-protocol analysis in the probiotic group vs the control group were 1.67 (95%CI: 1.38-2.02) and 1.68 (95%CI: 1.35-2.08), respectively, using the fixed-effects model. The sensitivity of the Asian studies was greater than that of the Caucasian studies (Asian: OR = 1.78, 95%CI: 1.40-2.26; Caucasian: OR = 1.48, 95%CI: 1.06-2.05). The pooled OR for the incidence of total adverse effects was significantly lower in the probiotic group (OR = 0.49, 95%CI: 0.26-0.94), using the random effects model, with significant heterogeneity (I2 = 85.7%). The incidence of diarrhea was significantly reduced in the probiotic group (OR = 0.21, 95%CI: 0.06-0.74), whereas the incidence of taste disorders, metallic taste, vomiting, nausea, and epigastric pain did not differ significantly between the probiotic group and the control group.
CONCLUSION: Supplementary probiotic preparations during standard triple H. pylori therapy may improve the eradication rate, particularly in Asian patients, and the incidence of total adverse effects.
doi:10.3748/wjg.v20.i47.18013
PMCID: PMC4273153  PMID: 25548501
Helicobacter pylori; Eradication; Probiotics; Meta-analysis; Adult
10.  Expression levels of matrix metalloproteinase-9 in human gastric carcinoma 
Oncology Letters  2014;9(2):915-919.
The present report investigated the correlation between the expression levels of matrix metalloproteinase (MMP)-9 in gastric carcinoma patients and the clinicopathological characteristics. Forty-five samples of gastric carcinoma and distal gastric mucosa tissue, and 10 samples of healthy gastric mucosa tissue were analyzed using semi-quantitative polymerase chain reaction, as well as immunohistochemical and hematoxylin and eosin staining. MMP-9 protein levels in serum samples from the same patients were quantified by enzyme-linked immunosorbent assay. The present report identified that MMP-9 expression was markedly higher in the gastric carcinoma tissue (86.67%) than in the adjacent healthy tissue (10.00%). A positive association was identified between the level of MMP-9 protein expression and the depth of cancer invasion (P<0.05). Furthermore, the preoperative serum levels of the MMP-9 protein in the gastric carcinoma tissue were correlated with the tumor-node-metastasis stage and occurrence of lymph node metastasis (P<0.01). Data from the present report indicates that MMP-9 may be key in gastric carcinoma malignancy, and implies that MMP-9 may serve as a novel biomarker in the diagnosis and prognosis of gastric carcinoma.
doi:10.3892/ol.2014.2768
PMCID: PMC4301519  PMID: 25621068
gastric carcinoma; matrix metalloproteinases-9; semi-quantitative polymerase chain reaction; immunohistochemistry; malignancy
11.  Characterization of a novel orthoreovirus isolated from fruit bat, China 
BMC Microbiology  2014;14(1):293.
Background
In recent years novel human respiratory disease agents have been described for Southeast Asia and Australia. The causative pathogens were classified as pteropine orthoreoviruses with a strong phylogenetic relationship to orthoreoviruses of bat origin.
Results
In this report, we isolated a novel Melaka-like reovirus (named “Cangyuan virus”) from intestinal content samples of one fruit bat residing in China’s Yunnan province. Phylogenetic analysis of the whole Cangyuan virus genome sequences of segments L, M and S demonstrated the genetic diversity of the Cangyuan virus. In contrast to the L and M segments, the phylogenetic trees for the S segments of Cangyuan virus demonstrated a greater degree of heterogeneity.
Conclusions
Phylogenetic analysis indicated that the Cangyuan virus was a novel orthoreovirus and substantially different from currently known members of Pteropine orthoreovirus (PRV) species group.
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-014-0293-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s12866-014-0293-4
PMCID: PMC4264558  PMID: 25433675
Bat Orthoreovirus; Prevalence; Viral genome reassortment
12.  Inhibition of Fumonisin B1 Cytotoxicity by Nanosilicate Platelets during Mouse Embryo Development 
PLoS ONE  2014;9(11):e112290.
Nanosilicate platelets (NSP), the form of natural silicate clay that was exfoliated from montmorillonite (MMT), is widely used as a feed additive for its high non-specific binding capacity with mycotoxins such as fumonisin B1 (FB1), and has been evaluated its safety for biomedical use including cytotoxicity, genotoxicity, and lethal dosage (LD). In the study, we further examined its toxicity on the development of CD1 mouse embryos and its capacity to prevent teratogenesis-induced by FB1. In vitro cultures, NSP did not disturb the development and the quality of intact pre-implantation mouse embryos. Further, newborn mice from females consumed with NSP showed no abnormalities. NSP had an unexpected high adsorption capacity in vitro. In contrast to female mice consumed with FB1 only, a very low residual level of FB1 in the circulation, reduced incidence of neutral tube defects and significantly increased fetal weight were observed in the females consumed with FB1 and NSP, suggesting a high alleviation effect of NSP on FB1 in vivo. Furthermore, FB1 treatment disturbed the gene expression of sphingolipid metabolism enzymes (longevity assurance homolog 5, LASS 5; sphingosine kinase 1, Sphk1; sphingosine kinase 2, Sphk2; sphingosine 1- phosphate lyase, Sgpl1; sphingosine 1-phosphate phosphatase, Sgpp1) in the maternal liver, uterus, fetus, and placenta, but NSP administration reversed the perturbations. Based on these findings, we conclude that NSP is a feasible and effective agent for supplementary use in reducing the toxicity of FB1 to animals.
doi:10.1371/journal.pone.0112290
PMCID: PMC4226500  PMID: 25383881
13.  Global View of the Functional Molecular Organization of the Avian Cerebrum: Mirror Images and Functional Columns 
The Journal of comparative neurology  2013;521(16):3614-3665.
Based on quantitative cluster analyses of 52 constitutively expressed or behaviorally regulated genes in 23 brain regions, we present a global view of telencephalic organization of birds. The patterns of constitutively expressed genes revealed a partial mirror image organization of three major cell populations that wrap above, around, and below the ventricle and adjacent lamina through the mesopallium. The patterns of behaviorally regulated genes revealed functional columns of activation across boundaries of these cell populations, reminiscent of columns through layers of the mammalian cortex. The avian functionally regulated columns were of two types: those above the ventricle and associated mesopallial lamina, formed by our revised dorsal mesopallium, hyperpallium, and intercalated hyperpallium; and those below the ventricle, formed by our revised ventral mesopallium, nidopallium, and intercalated nidopallium. Based on these findings and known connectivity, we propose that the avian pallium has four major cell populations similar to those in mammalian cortex and some parts of the amygdala: 1) a primary sensory input population (intercalated pallium); 2) a secondary intrapallial population (nidopallium/hyperpallium); 3) a tertiary intrapallial population (mesopallium); and 4) a quaternary output population (the arcopallium). Each population contributes portions to columns that control different sensory or motor systems. We suggest that this organization of cell groups forms by expansion of contiguous developmental cell domains that wrap around the lateral ventricle and its extension through the middle of the mesopallium. We believe that the position of the lateral ventricle and its associated mesopallium lamina has resulted in a conceptual barrier to recognizing related cell groups across its border, thereby confounding our understanding of homologies with mammals.
doi:10.1002/cne.23404
PMCID: PMC4145244  PMID: 23818122
forebrain; brain pathways; brain organization; neural activity; motor behavior; primary sensory; brain evolution; neurotransmitter receptors; immediate early genes; pallium; cortex; striatum; pallidum; basal ganglia; amygdala; claustrum
14.  Efficient non-doped phosphorescent orange, blue and white organic light-emitting devices 
Scientific Reports  2014;4:6754.
Efficient phosphorescent orange, blue and white organic light-emitting devices (OLEDs) with non-doped emissive layers were successfully fabricated. Conventional blue phosphorescent emitters bis [4,6-di-fluorophenyl]-pyridinato-N,C2′] picolinate (Firpic) and Bis(2,4-difluorophenylpyridinato) (Fir6) were adopted to fabricate non-doped blue OLEDs, which exhibited maximum current efficiency of 7.6 and 4.6 cd/A for Firpic and Fir6 based devices, respectively. Non-doped orange OLED was fabricated utilizing the newly reported phosphorescent material iridium (III) (pbi)2Ir(biq), of which manifested maximum current and power efficiency of 8.2 cd/A and 7.8 lm/W. The non-doped white OLEDs were achieved by simply combining Firpic or Fir6 with a 2-nm (pbi)2Ir(biq). The maximum current and power efficiency of the Firpic and (pbi)2Ir(biq) based white OLED were 14.8 cd/A and 17.9 lm/W.
doi:10.1038/srep06754
PMCID: PMC4208040  PMID: 25341698
15.  Mammary Fat of Breast Cancer: Gene Expression Profiling and Functional Characterization 
PLoS ONE  2014;9(10):e109742.
Mammary fat is the main composition of breast, and is the most probable candidate to affect tumor behavior because the fat produces hormones, growth factors and adipokines, a heterogeneous group of signaling molecules. Gene expression profiling and functional characterization of mammary fat in Chinese women has not been reported. Thus, we collected the mammary fat tissues adjacent to breast tumors from 60 subjects, among which 30 subjects had breast cancer and 30 had benign lesions. We isolated and cultured the stromal vascular cell fraction from mammary fat. The expression of genes related to adipose function (including adipogenesis and secretion) was detected at both the tissue and the cellular level. We also studied mammary fat browning. The results indicated that fat tissue close to malignant and benign lesions exhibited distinctive gene expression profiles and functional characteristics. Although the mammary fat of breast tumors atrophied, it secreted tumor growth stimulatory factors. Browning of mammary fat was observed and browning activity of fat close to malignant breast tumors was greater than that close to benign lesions. Understanding the diversity between these two fat depots may possibly help us improve our understanding of breast cancer pathogenesis and find the key to unlock new anticancer therapies.
doi:10.1371/journal.pone.0109742
PMCID: PMC4188628  PMID: 25291184
16.  Experimental Comparison of the Reproductive Outcomes and Early Development of the Offspring of Rats Given Five Common Types of Drinking Water 
PLoS ONE  2014;9(10):e108955.
Tap water (unfiltered), filtered tap water and processed bottled water (purified water, artificial mineralized water, or natural water) are now the five most widely consumed types of drinking water in China. However, the constituents (organic chemicals and inorganic ingredients) of the five waters differ, which may cause them to have different long-term health effects on those who drink them, especially sensitive children. In order to determine which type of water among the five waters is the most beneficial regarding reproductive outcomes and the developmental behaviors of offspring, two generations of Sprague–Dawley rats were given these five waters separately, and their reproductive outcomes and the developmental behaviors of their offspring were observed and compared. The results showed that the unfiltered tap water group had the lowest values for the maternal gestation index (MGI) and offspring's learning and memory abilities (OLMA); the lowest offspring survival rate was found in the purified water group; and the highest OLMA were found in the filtered tap water group. Thus, the best reproductive and offspring early developmental outcomes were found in the group that drank filtered tap water, which had the lowest levels of pollutants and the richest minerals. Therefore, thoroughly removing toxic contaminants and retaining the beneficial minerals in drinking water may be important for both pregnant women and children, and the best way to treat water may be with granular activated carbon and ion exchange by copper zinc alloy.
doi:10.1371/journal.pone.0108955
PMCID: PMC4184831  PMID: 25279561
17.  Steady-State Equilibrium Phase Inversion Recovery ON-resonant Water Suppression (IRON) Magnetic Resonance Angiography in Conjunction with Superparamagnetic Nanoparticles. A Robust Technique for Imaging within a Wide Range of Contrast Agent Dosages 
Objectives
To investigate the ability of inversion recovery ON-resonant water suppression (IRON) in conjunction with P904 (superparamagnetic nanoparticles which consisting of a maghemite core coated with a low-molecular-weight amino-alcohol derivative of glucose) to perform steady-state equilibrium phase magnetic resonance angiography (MRA) over a wide dose range.
Materials and Methods
Experiments were approved by the institutional animal care committee. Rabbits (n=12) were imaged at baseline and serially after the administration of 10 incremental dosages of 0.57–5.7 mgFe/Kg P904. Conventional T1-weighted and IRON MRA were obtained on a clinical 1.5-T scanner to image the thoracic and abdominal aorta, and peripheral vessels. Contrast-to-noise ratios (CNR) and vessel sharpness were quantified.
Results
Using IRON MRA, CNR and vessel sharpness progressively increased with incremental dosages of the contrast agent P904, exhibiting constantly higher contrast values than T1-weighted MRA over a very wide range of contrast agent doses (CNR of 18.8±5.6 for IRON versus 11.1±2.8 for T1-weighted MRA at 1.71 mgFe/kg, p=0.02 and 19.8±5.9 for IRON versus −0.8±1.4 for T1-weighted MRA at 3.99 mgFe/kg, p=0.0002). Similar results were obtained for vessel sharpness in peripheral vessels, (Vessel sharpness of 46.76±6.48% for IRON versus 33.20±3.53% for T1-weighted MRA at 1.71 mgFe/Kg, p=0.002, and of 48.66±5.50% for IRON versus 19.00±7.41% for T1-weighted MRA at 3.99 mgFe/Kg, p=0.003).
Conclusion
Our study suggests that quantitative CNR and vessel sharpness after the injection of P904 are consistently higher for IRON MRA when compared to conventional T1-weighted MRA. These findings apply for a wide range of contrast agent dosages.
doi:10.1002/jmri.24043
PMCID: PMC3657577  PMID: 23418107
18.  Relationship between homocysteine level and diabetic retinopathy: a systematic review and meta-analysis 
Diagnostic Pathology  2014;9(1):167.
Background
The relationship between homocysteine (Hcy) and diabetic retinopathy (DR) remains unclear to date. Therefore, a systematic review and meta-analysis was performed on the relationship between Hcy level and DR.
Methods
Studies were identified by searching PubMed, Embase, and Web of Science databases until 5 May, 2014.
Results
A total of 31 studies involving 6,394 participants were included in the meta-analysis. After pooling the data from each included study, the blood Hcy concentration in the DR group was observed to be higher than that in the control group [WMD = 2.55; 95% confidence interval (CI), 1.70–3.40], and diabetes mellitus (DM) patients with hyperhomocysteinemia were at a risk for DR [odds ratio (OR) = 1.93; 95% CI, 1.46–2.53]. Considering the different DM types, hyperhomocysteinemia in T1DM (OR = 1.83, 95% CI, 1.28–2.62) was associated with DR rather than in T2DM (OR = 1.59, 95% CI, 0.72–3.51). Considerable statistical heterogeneity in the overall summary estimates was partly explained by the geographical differences.
Conclusions
Results from this current meta-analysis indicate that hyperhomocysteinemia is a risk factor for DR, especially proliferative DR. Differences between geographical regions were observed in the relationship between hyperhomocysteinemia with T1DM risk. Given the heterogeneous results, the relationship between high Hcy and DR needs further investigation.
Virtual Slides
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_167
doi:10.1186/s13000-014-0167-y
PMCID: PMC4207897  PMID: 25257241
Hyperhomocysteinemia; Homocysteine; Diabetic retinopathy (DR)
19.  Mitochondrial ferritin, a new target for inhibiting neuronal tumor cell proliferation 
Mitochondrial ferritin (FtMt) has a significant effect on the regulation of cytosolic and mitochondrial iron levels. However, because of the deficiency of iron regulatory elements (IRE) in FtMt’s gene sequence, the exact function of FtMt remains unclear. In the present study, we found that FtMt dramatically inhibited SH-SY5Y cell proliferation and tumor growth in nude mice. Interestingly, excess FtMt did not adversely affect the development of drosophila. Additionally, we found that the expression of FtMt in human normal brain tissue was significantly higher than that of neuroblastoma, but not higher than that of neurospongioma. However, the expression of transferrin receptor 1 is completely opposite. We therefore hypothesized that increased expression of FtMt may negatively affect the vitality of neuronal tumor cells. Therefore, we further investigated the underlying mechanisms of FtMt’s inhibitory effects on neuronal tumor cell proliferation. As expected, FtMt overexpression disturbed the iron homeostasis of tumor cells and significantly downregulated the expression of proliferating cell nuclear antigen. Moreover, FtMt affected cell cycle, causing G1/S arrest by modifying the expression of cyclinD1, cyclinE, Cdk2, Cdk4 and p21. Remarkably, FtMt strongly upregulated the expression of the tumor suppressors, p53 and N-myc downstream-regulated gene-1 (NDRG1), but dramatically decreased C-myc, N-myc and p-Rb levels. This study demonstrates for the first time a new role and mechanism for FtMt in the regulation of cell cycle. We thus propose FtMt as a new candidate target for inhibiting neuronal tumor cell proliferation. Appropriate regulation of FtMt expression may prevent tumor cell growth. Our study may provide a new strategy for neuronal cancer therapy.
Electronic supplementary material
The online version of this article (doi:10.1007/s00018-014-1730-0) contains supplementary material, which is available to authorized users.
doi:10.1007/s00018-014-1730-0
PMCID: PMC4323545  PMID: 25213357
Neuroblastoma; Cyclin; Cell cycle; Cyclin-dependent protein kinase; Iron metabolism
20.  Direct in vitro comparison of six 3D positive contrast methods for susceptibility marker imaging 
Purpose
To compare different techniques for positive contrast imaging of susceptibility markers with MRI for 3D visualization. As several different techniques have been reported, the choice of the suitable method depends on its properties with regard to the amount of positive contrast and the desired background suppression, as well as other imaging constraints needed for a specific application.
Materials and methods
Six different positive contrast techniques are investigated for their ability to image at 3T a single susceptibility marker in vitro. The white marker method (WM), susceptibility gradient mapping (SGM), inversion recovery with on-resonant water suppression (IRON), frequency selective excitation (FSX), fast low flip-angle positive contrast SSFP (FLAPS), and iterative decomposition of water and fat with echo asymmetry and least-squares estimation (IDEAL) were implemented and investigated.
Results
The different methods were compared with respect to the volume of positive contrast, the product of volume and signal intensity, imaging time, and the level of background suppression. Quantitative results are provided and strengths and weaknesses of the different approaches are discussed.
Conclusion
The appropriate choice of positive contrast imaging technique depends on the desired level of background suppression, acquisition speed, and robustness against artifacts, for which in vitro comparative data is now available.
doi:10.1002/jmri.23976
PMCID: PMC3620818  PMID: 23281151
susceptibility imaging; off resonance; positive contrast
21.  The Extracellular Microenvironment Explains Variations in Passive Drug Transport across Different Airway Epithelial Cell Types 
Pharmaceutical research  2013;30(8):2118-2132.
Purpose
We sought to identify key variables in cellular architecture and physiology that might explain observed differences in the passive transport properties of small molecule drugs across different airway epithelial cell types.
Methods
Propranolol (PR) was selected as a weakly basic, model compound to compare the transport properties of primary (NHBE) vs. tumor-derived (Calu-3) cells. Differentiated on Transwell™ inserts, the architecture of pure vs. mixed cell co-cultures was studied with confocal microscopy followed by quantitative morphometric analysis. Cellular pharmacokinetic modeling was used to identify parameters that differentially affect PR uptake and transport across these two cell types.
Results
Pure Calu-3 and NHBE cells possessed different structural and functional properties. Nevertheless, mixed Calu-3 and NHBE cell co-cultures differentiated as stable cell monolayers. After measuring the total mass of PR, the fractional areas covered by Calu-3 and NHBE cells allowed deconvoluting the transport properties of each cell type. Based on the apparent thickness of the unstirred, cell surface aqueous layer, local differences in extracellular microenvironment explained the measured variations in passive PR uptake and permeation between Calu-3 and NHBE cells.
Conclusion
Mixed cell co-cultures can be used to compare the local effects of the extracellular microenvironment on drug uptake and transport across two epithelial cell types.
doi:10.1007/s11095-013-1069-5
PMCID: PMC3706189  PMID: 23708857
cellular pharmacokinetics; Calu-3 cells; local drug absorption; inhaled drug delivery; computational modeling
22.  Arabidopsis LIP5, a Positive Regulator of Multivesicular Body Biogenesis, Is a Critical Target of Pathogen-Responsive MAPK Cascade in Plant Basal Defense 
PLoS Pathogens  2014;10(7):e1004243.
Multivesicular bodies (MVBs) play essential roles in many cellular processes. The MVB pathway requires reversible membrane association of the endosomal sorting complexes required for transports (ESCRTs) for sustained protein trafficking. Membrane dissociation of ESCRTs is catalyzed by the AAA ATPase SKD1, which is stimulated by LYST-INTERACTING PROTEIN 5 (LIP5). We report here that LIP5 is a target of pathogen-responsive mitogen-activated protein kinases (MPKs) and plays a critical role in plant basal resistance. Arabidopsis LIP5 interacts with MPK6 and MPK3 and is phosphorylated in vitro by activated MPK3 and MPK6 and in vivo upon expression of MPK3/6-activating NtMEK2DD and pathogen infection. Disruption of LIP5 has little effects on flg22-, salicylic acid-induced defense responses but compromises basal resistance to Pseudomonas syringae. The critical role of LIP5 in plant basal resistance is dependent on its ability to interact with SKD1. Mutation of MPK phosphorylation sites in LIP5 does not affect interaction with SKD1 but reduces the stability and compromises the ability to complement the lip5 mutant phenotypes. Using the membrane-selective FM1–43 dye and transmission electron microscopy, we demonstrated that pathogen infection increases formation of both intracellular MVBs and exosome-like paramural vesicles situated between the plasma membrane and the cell wall in a largely LIP5-dependent manner. These results indicate that the MVB pathway is positively regulated by pathogen-responsive MPK3/6 through LIP5 phosphorylation and plays a critical role in plant immune system likely through relocalization of defense-related molecules.
Author Summary
Pathogen- and stress-responsive mitogen-activated protein kinases 3 and 6 (MPK3/6) cascade plays an important role in plant basal resistance to microbial pathogens. Here we showed that Arabidopsis MPK3 and MPK6 interact with and phosphorylate the LIP5 positive regulator of biogenesis of multivesicular bodies (MVBs), which are unique organelles containing small vesicles in their lumen. Disruption of LIP5 causes increased susceptibility to the bacterial pathogen Pseudomonas syringae. Compromised disease resistance of the lip5 mutants is associated with competent flg22- and salicylic acid-induced defense responses but compromised accumulation of intracellular MVBs and exosome-like paramural vesicles, which have previously been shown to be involved in the relocalization of defense-related molecules. Phosphorylation by MPK3/6 increases LIP5 stability, which is necessary for pathogen-induced MVB trafficking and basal disease resistance. Based on these results we conclude that the MVB pathway is positively regulated by pathogen-responsive MPK3/6 through LIP5 phosphorylation and plays a critical role in plant immune system probably through involvement in the relocalization of defense-related molecules.
doi:10.1371/journal.ppat.1004243
PMCID: PMC4092137  PMID: 25010425
23.  Facilitating Cytokine-Mediated Cancer Cell Death by Proteobacterial N-Acylhomoserine Lactones 
ACS chemical biology  2013;8(6):1117-1120.
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) preferentially induces apoptosis in cancer cells over normal cells; however, tumor cells may develop TRAIL resistance. Here we demonstrate that this resistance can be overcome in the presence of bacterial acylhomoserine lactones (AHLs) or AHL-producing bacteria through the combined effect of TRAIL-induced apoptosis and AHL-mediated inhibition of inflammation regulated by NF-κB signaling. This discovery unveils a previously unrecognized symbiotic link between bacteria and host immunosurveillance.
doi:10.1021/cb4000184
PMCID: PMC3755046  PMID: 23517377
24.  Molecular mechanism of fluoroquinolones resistance in Mycoplasma hominis clinical isolates 
Brazilian Journal of Microbiology  2014;45(1):239-242.
To evaluate the molecular mechanism of fluoroquinolones resistance in Mycoplasma hominis (MH) clinical strains isolated from urogenital specimens. 15 MH clinical isolates with different phenotypes of resistance to fluoroquinolones antibiotics were screened for mutations in the quinolone resistance-determining regions (QRDRs) of DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) in comparison with the reference strain PG21, which is susceptible to fluoroquinolones antibiotics. 15 MH isolates with three kinds of quinolone resistance phenotypes were obtained. Thirteen out of these quinolone-resistant isolates were found to carry nucleotide substitutions in either gyrA or parC. There were no alterations in gyrB and no mutations were found in the isolates with a phenotype of resistance to Ofloxacin (OFX), intermediate resistant to Levofloxacin (LVX) and Sparfloxacin (SFX), and those susceptible to all three tested antibiotics. The molecular mechanism of fluoroquinolone resistance in clinical isolates of MH was reported in this study. The single amino acid mutation in ParC of MH may relate to the resistance to OFX and LVX and the high-level resistance to fluoroquinolones for MH is likely associated with mutations in both DNA gyrase and the ParC subunit of topoisomerase IV.
PMCID: PMC4059304  PMID: 24948939
Mycoplasma hominis; quinolones; drug resistance; mutation
25.  Phosphopeptide Enrichment with TiO2-Modified Membranes and Investigation of Tau Protein Phosphorylation 
Analytical chemistry  2013;85(12):5699-5706.
Selective enrichment of phosphopeptides prior to their analysis by mass spectrometry (MS) is vital for identifying protein phosphorylation sites involved in cellular regulation. This study describes modification of porous nylon substrates with TiO2 nanoparticles to create membranes that rapidly enrich phosphopeptides. Membranes with a 22-mm diameter bind 540 nmol of phosphoangiotensin and recover 70% of the phosphopeptides in mixtures with a 15-fold excess of non-phosphorylated proteins. Recovery is 90% for a pure phosphopeptide. Insertion of small membrane disks into HPLC fittings allows rapid enrichment of 5 mL of 1 fmol/μL phosphoprotein digests and concentration into small-volume (10’s of μL) eluates. The combination of membrane enrichment with tandem mass spectrometry reveals seven phosphorylation sites from in vivo phosphorylated tau (p-tau) protein, which is associated with Alzheimer’s disease.
doi:10.1021/ac400198n
PMCID: PMC3721342  PMID: 23638980

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