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1.  Distribution of iron- and sulfate-reducing bacteria across a coastal acid sulfate soil (CASS) environment: implications for passive bioremediation by tidal inundation 
Coastal acid sulfate soils (CASS) constitute a serious and global environmental problem. Oxidation of iron sulfide minerals exposed to air generates sulfuric acid with consequently negative impacts on coastal and estuarine ecosystems. Tidal inundation represents one current treatment strategy for CASS, with the aim of neutralizing acidity by triggering microbial iron- and sulfate-reduction and inducing the precipitation of iron-sulfides. Although well-known functional guilds of bacteria drive these processes, their distributions within CASS environments, as well as their relationships to tidal cycling and the availability of nutrients and electron acceptors, are poorly understood. These factors will determine the long-term efficacy of “passive” CASS remediation strategies. Here we studied microbial community structure and functional guild distribution in sediment cores obtained from 10 depths ranging from 0 to 20 cm in three sites located in the supra-, inter- and sub-tidal segments, respectively, of a CASS-affected salt marsh (East Trinity, Cairns, Australia). Whole community 16S rRNA gene diversity within each site was assessed by 454 pyrotag sequencing and bioinformatic analyses in the context of local hydrological, geochemical, and lithological factors. The results illustrate spatial overlap, or close association, of iron-, and sulfate-reducing bacteria (SRB) in an environment rich in organic matter and controlled by parameters such as acidity, redox potential, degree of water saturation, and mineralization. The observed spatial distribution implies the need for empirical understanding of the timing, relative to tidal cycling, of various terminal electron-accepting processes that control acid generation and biogeochemical iron and sulfur cycling.
PMCID: PMC4490247  PMID: 26191042
acid sulfate; microbial community; iron-reducing; sulfate-reducing; iron sulfides; geomicrobiology; wetlands; remediation
2.  Are Young Children with Cochlear Implants Sensitive to the Statistics of Words in the Ambient Spoken Language? 
To determine whether children with cochlear implants (CIs) are sensitive to statistical characteristics of words in the ambient spoken language, whether that sensitivity changes in expected ways as their spoken lexicon grows, and whether that sensitivity varies with unilateral or bilateral implantation.
We analyzed archival data collected from the parents of 36 children who received cochlear implantation (20 unilateral, 16 bilateral) before 24 months of age. The parents reported their children’s word productions 12 months after implantation using the MacArthur Communicative Development Inventories: Words and Sentences (Fenson et al., 1993). We computed the number of words, out of 292 possible monosyllabic nouns, verbs, and adjectives, that each child was reported to say and calculated the average phonotactic probability, neighborhood density, and word frequency of the reported words.
Spoken vocabulary size positively correlated with average phonotactic probability and negatively correlated with average neighborhood density, but only in children with bilateral CIs.
At 12 months post-implantation, children with bilateral CIs demonstrate sensitivity to statistical characteristics of words in the ambient spoken language akin to that reported for hearing children during the early stages of lexical development. Children with unilateral CIs do not.
PMCID: PMC4490103  PMID: 25677929
Cochlear implants; children; phonotactic probability; neighborhood density; word frequency; emerging lexicon
3.  KDM4B as a Target for Prostate Cancer: Structural Analysis and Selective Inhibition by a Novel Inhibitor 
Journal of Medicinal Chemistry  2014;57(14):5975-5985.
The KDM4/JMJD2 Jumonji C-containing histone lysine demethylases (KDM4A–KDM4D), which selectively remove the methyl group(s) from tri/dimethylated lysine 9/36 of H3, modulate transcriptional activation and genome stability. The overexpression of KDM4A/KDM4B in prostate cancer and their association with androgen receptor suggest that KDM4A/KDM4B are potential progression factors for prostate cancer. Here, we report the crystal structure of the KDM4B·pyridine 2,4-dicarboxylic acid·H3K9me3 ternary complex, revealing the core active-site region and a selective K9/K36 site. A selective KDM4A/KDM4B inhibitor, 4, that occupies three subsites in the binding pocket is identified by virtual screening. Pharmacological and genetic inhibition of KDM4A/KDM4B significantly blocks the viability of cultured prostate cancer cells, which is accompanied by increased H3K9me3 staining and transcriptional silencing of growth-related genes. Significantly, a substantial portion of differentially expressed genes are AR-responsive, consistent with the roles of KDM4s as critical AR activators. Our results point to KDM4 as a useful therapeutic target and identify a new inhibitor scaffold.
PMCID: PMC4216216  PMID: 24971742
4.  Prognostic Model Based on Systemic Inflammatory Response and Clinicopathological Factors to Predict Outcome of Patients with Node-Negative Gastric Cancer 
PLoS ONE  2015;10(6):e0128540.
Prognostic models are generally used to predict gastric cancer outcomes. However, no model combining patient-, tumor- and host-related factors has been established to predict outcomes after radical gastrectomy, especially outcomes of patients without nodal involvement. The aim of this study was to develop a prognostic model based on the systemic inflammatory response and clinicopathological factors of resectable gastric cancer and determine whether the model can improve prognostic accuracy in node-negative patients. We reviewed the clinical, laboratory, histopathological and survival data of 1397 patients who underwent radical gastrectomy between 2007 and 2013. Patients were split into development and validation sets of 1123 and 274 patients, respectively. Among all 1397 patients, 545 had node-negative gastric cancer; 440 were included in the development set, 105 were included in the validation set. A prognostic model was constructed from the development set. The scoring system was based on hazard ratios in a Cox proportional hazard model. In the multivariate analysis, age, tumor size, Lauren type, depth of invasion, lymph node metastasis, and the neutrophil—lymphocyte ratio were independent prognostic indicators of overall survival. A prognostic model was then established based on the significant factors. Patients were categorized into five groups according to their scores. The 3-year survival rates for the low- to high-risk groups were 98.9%, 92.8%, 82.4%, 58.4%, and 36.9%, respectively (P < 0.001). The prognostic model clearly discriminated patients with stage pT1-4N0M0 tumor into four risk groups with significant differences in the 3-year survival rates (P < 0.001). Compared with the pathological T stage, the model improved the predictive accuracy of the 3-year survival rate by 5% for node-negative patients. The prognostic scores also stratified the patients with stage pT4aN0M0 tumor into significantly different risk groups (P = 0.004). Furthermore, the predictive value of this model was validated in an independent set of 274 patients. This model, which included the systemic inflammatory markers and clinicopathological factors, is more effective in predicting the prognosis of node-negative gastric cancer than traditional staging systems. Patients in the high-risk group might be good candidates for adjuvant chemotherapy.
PMCID: PMC4468084  PMID: 26075713
5.  Association of assisted reproductive technology with adverse pregnancy outcomes 
More and more infertile patients have accepted the assisted reproductive technique (ART) therapy. Concerns have been raised over an increased risk of adverse maternal outcomes in ART populations as compared with natural conception (NC).
The aim was to improve the ART in clinicial work and to reduce the incidence of pregnancy complications in ART group according to analyzing the reasons of high incidence of pregnancy complications in ART group, comparing the incidence of pregnancy complications in different controlled ovarian hyperstimulation (COH) programs and evaluating the effects of ART which attribute to adverse pregnancy outcomes.
Materials and Methods:
In this prospective population-based cohort study,3216 pregnant women with gestational age ≤12 weeks, regular antenatal examination,and ultrasound identification of intrauterine pregnancy were enrolled from January 2010 to June 2013. According to having ART history, the participantswere divided into two groups: ART group (contains fresh embryo transfer group or frozen-thawed embryo transfer group) and NC group. We compared the incidence of pregnancy complications between different groups and evaluated the factors which could affect the occurrence of these complications.
When compared to NC group, significantly increased rates of gestational diabetes mellitus (GDM) (p<0.01), preeclampsia (PE) (p<0.01) and intrahepatic cholestasis of pregnancy (ICP) (p˂0.01) were observed in ART group. There was no significant difference in the incidence of birth defect between the two groups (p=0.07). Multiple pregnancies and Gonadotropin (Gn) were risk factors in GDM, PE, and ICP. The exogenous progesterone treatment had no effect on GDM, PE or ICP.
ART increases the risk of adverse maternal complications such as GDM, PE and ICP. The dosages of Gn should be reduced to an extent and the number of embryo implantation should be controlled. Exogenous progesterone treatment is safe.
PMCID: PMC4426157  PMID: 26000008
Infertility; Assisted reproductive technique; Pregnancy complications
6.  Lentivirus-mediated RNAi knockdown of the gap junction protein, Cx43, attenuates the development of vascular restenosis following balloon injury 
Percutaneous coronary intervention [PCI or percutaneous transluminal coronary angioplasty (PTCA)] has been developed into a mature interventional treatment for atherosclerotic cardiovascular disease. However, the long-term therapeutic effect is compromised by the high incidence of vascular restenosis following angioplasty, and the underlying mechanisms of vascular restenosis have not yet been fully elucidated. In the present study, we investigated the role of the gap junction (GJ) protein, connexin 43 (Cx43), in the development of vascular restenosis. To establish vascular restenosis, rat carotid arteries were subjected to balloon angioplasty injury. At 0, 7, 14 and 2 days following balloon injury, the arteries were removed, and the intimal/medial area of the vessels was measured to evaluate the degree of restenosis. We found that the intimal area gradually increased following balloon injury. Intimal hyperplasia and restenosis were particularly evident at 14 and 28 days after injury. In addition, the mRNA and protein expression of Cx43 was temporarily decreased at 7 days, and subsequently increased at 14 and 28 days following balloon injury, as shown by RT-PCR and western blot analysis. To determine the involvement of Cx43 in vascular restenosis, the lentivirus vector expressing shRNA targeting Cx43, Cx43-RNAi-LV, was used to silence Cx43 in the rat carotid arteries. The knockdown of Cx43 effectively attenuated the development of intimal hyperplasia and vascular restenosis following balloon injury. Thus, our data indicate the vital role of the GJ protein, Cx43, in the development of vascular restenosis, and provide new insight into the pathogenesis of vascular reste-nosis. Cx43 may prove to be a novel potential pharmacological target for the prevention of vascular restenosis following PCI.
PMCID: PMC4356439  PMID: 25625334
gap junction; connexin 43; balloon injury; vascular restenosis; RNA interference
7.  IL1RAP as a surface marker for leukemia stem cells is related to clinical phase of chronic myeloid leukemia patients 
Chronic myeloid leukemia (CML) is a clonal disease from hematopoietic stem cells. Surviving leukemia stem cells (LSCs) and progenitor cells are a potential source for CML relapse and progression. Recent data reported that IL-1 receptor accessory protein (IL1RAP) gene was differentially expressed in CML versus normal stem and progenitor cells. However, whether the level of IL1RAP is associated with clinical phases of CML, and correlations between IL1RAP expression and detections of diagnosis is still unclear. Here we demonstrated that IL1RAP was up-regulated in CD34+ and CD34+CD38- cells which highly enriched with stem cells. Furthermore, IL1RAP expression in CD34+CD38- cells was tightly consistent with the generation of BCR-ABL fusion gene and Philadelphia chromosome. Importantly, we found that the level of IL1RAP increased with disease progression from chronic phase (CP) into accelerated phase (AP) and blast phase (BP), which was investigated not only in new diagnosed CML patients but also in patients treated with tyrosine kinase inhibitors (TKI) and hydroxyurea. Negative correlation was detected between IL1RAP expression and neutrophil (NE), whereas no relation was found in white blood cell (WBC), lymphocyte (LY), red blood cell (RBC), platelet (PLT), age or gender of CML patients. In conclusion, we identified IL1RAP as a surface marker of LSCs may be a potential indicator for CML clinical phases.
PMCID: PMC4307422  PMID: 25663975
Chronic myeloid leukemia; leukemia stem cells; IL-1 receptor accessory protein
8.  Absence of Gamma-Interferon-Inducible Lysosomal Thiol Reductase (GILT) Is Associated with Poor Disease-Free Survival in Breast Cancer Patients 
PLoS ONE  2014;9(10):e109449.
Tumor immunosurveillance is known to be of critical importance in controlling tumorigenesis and progression in various cancers. The role of gamma-interferon-inducible lysosomal thiol reductase (GILT) in tumor immunosurveillance has recently been studied in several malignant diseases, but its role in breast cancer remains to be elucidated. In the present study, we found GILT as a significant different expressed gene by cDNA microarray analysis. To further determine the role of GILT in breast cancer, we examined GILT expression in breast cancers as well as noncancerous breast tissues by immunohistochemistry and real-time PCR, and assessed its association with clinicopathologic characteristics and patient outcome. The absence of GILT expression increased significantly from 2.02% (2/99) in noncancerous breast tissues to 15.6% (34/218) in breast cancer tissues (P<0.001). In accordance with its proliferation inhibiting function, GILT expression was inversely correlated with Ki67 index (P<0.05). In addition, absence of GILT was positively correlated with adverse characteristics of breast cancers, such as histological type, tumor size, lymph nodes status, and pTNM stage (P<0.05). Consistently, breast cancers with reduced GILT expression had poorer disease-free survival (P<0.005). Moreover, significantly decreased expression of GILT was found in both primary and metastatic breast cancer cells, in contrast to normal epithelial cells. These findings indicate that GILT may act as a tumor suppressor in breast cancer, in line with its previously suggested role in anti-tumor immunity. Thus, GILT has the potential to be a novel independent prognostic factor in breast cancer and further studies are needed to illustrate the underlying mechanism of this relationship.
PMCID: PMC4204821  PMID: 25333930
9.  Sensitization of neurons in the central nucleus of the amygdala via the decreased GABAergic inhibition contributes to the development of neuropathic pain-related anxiety-like behaviors in rats 
Molecular Brain  2014;7:72.
Despite high prevalence of anxiety accompanying with chronic pain, the mechanisms underlying pain-related anxiety are largely unknown. With its well-documented role in pain and emotion processing, the amygdala may act as a key player in pathogenesis of neuropathic pain-related anxiety. Pain-related plasticity and sensitization of CeA (central nucleus of the amygdala) neurons have been shown in several models of chronic pain. In addition, firing pattern of neurons with spike output can powerfully affect functional output of the brain nucleus, and GABAergic neurons are crucial in the modulation of neuronal excitability. In this study, we first investigated whether pain-related plasticity (e.g. alteration of neuronal firing patterns) and sensitization of CeA neurons contribute to nerve injury-evoked anxiety in neuropathic rats. Furthermore, we explored whether GABAergic disinhibition is responsible for regulating firing patterns and intrinsic excitabilities of CeA neurons as well as for pain-related anxiety in neuropathic rats.
We discovered that spinal nerve ligation (SNL) produced neuropathic pain-related anxiety-like behaviors in rats, which could be specifically inhibited by intra-CeA administration of anti-anxiety drug diazepam. Moreover, we found potentiated plasticity and sensitization of CeA neurons in SNL-induced anxiety rats, of which including: 1) increased burst firing pattern and early-adapting firing pattern; 2) increased spike frequency and intrinsic excitability; 3) increased amplitude of both after-depolarized-potential (ADP) and sub-threshold membrane potential oscillation. In addition, we observed a remarkable reduction of GABAergic inhibition in CeA neurons in SNL-induced anxiety rats, which was proved to be important for altered firing patterns and hyperexcitability of CeA neurons, thereby greatly contributing to the development of neuropathic pain-related anxiety. Accordantly, activation of GABAergic inhibition by intra-CeA administration of muscimol, a selective GABAA receptors agonist, could inhibit SNL-induced anxiety-like behaviors in neuropathic rats. By contrast, suppression of GABAergic inhibition by intra-CeA administration of bicuculline, a selective GABAA receptors antagonist, produced anxiety-like behavior in normal rats.
This study suggests that reduction of GABAergic inhibition may be responsible for potentiated plasticity and sensitization of CeA neurons, which likely underlie the enhanced output of amygdala and neuropathic pain-related anxiety in SNL rats.
Electronic supplementary material
The online version of this article (doi:10.1186/s13041-014-0072-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4201706  PMID: 25277376
Anxiety; Neuropathic pain; Firing pattern; CeA; GABA
10.  Exercise Preconditioning Protects against Spinal Cord Injury in Rats by Upregulating Neuronal and Astroglial Heat Shock Protein 72 
The heat shock protein 72 (HSP 72) is a universal marker of stress protein whose expression can be induced by physical exercise. Here we report that, in a localized model of spinal cord injury (SCI), exercised rats (given pre-SCI exercise) had significantly higher levels of neuronal and astroglial HSP 72, a lower functional deficit, fewer spinal cord contusions, and fewer apoptotic cells than did non-exercised rats. pSUPER plasmid expressing HSP 72 small interfering RNA (SiRNA-HSP 72) was injected into the injured spinal cords. In addition to reducing neuronal and astroglial HSP 72, the (SiRNA-HSP 72) significantly attenuated the beneficial effects of exercise preconditioning in reducing functional deficits as well as spinal cord contusion and apoptosis. Because exercise preconditioning induces increased neuronal and astroglial levels of HSP 72 in the gray matter of normal spinal cord tissue, exercise preconditioning promoted functional recovery in rats after SCI by upregulating neuronal and astroglial HSP 72 in the gray matter of the injured spinal cord. We reveal an important function of neuronal and astroglial HSP 72 in protecting neuronal and astroglial apoptosis in the injured spinal cord. We conclude that HSP 72-mediated exercise preconditioning is a promising strategy for facilitating functional recovery from SCI.
PMCID: PMC4227258  PMID: 25334068
spinal cord compression; neurological severity score; heat shock protein 72; exercise preconditioning
11.  Stromal responses among common carcinomas correlated with clinicopathologic features 
We have previously characterized a tumor stroma expression signature in a subset of breast tumors that correlates with better clinical outcome. The purpose of this study is to determine whether this stromal signature, termed the ‘DTF fibroblast’ signature, is specific to breast cancer or is a common stromal response found in different types of cancer.
Experimental Designs
The DTF fibroblast signature was applied to gene expression profiles from five ovarian, five lung, two colon and three prostate cancer expression microarray datasets. Additionally, two different tissue microarrays of 204 ovarian tumors and 140 colon tumors were examined for the expression of previously characterized protein markers of DTF fibroblast signature. The DTF fibroblast stromal response was then correlated with clinicopathologic features.
The DTF fibroblast signature is robustly present in ovarian, lung, and colon carcinomas. Both expression microarray data and immunohistochemistry show that the subset of ovarian tumors with strong DTF fibroblast signature expression has statistically significant worse survival outcomes. No reproducible survival differences were found in either the lung or the colon cancers. The prostate cancers failed to demonstrate a DTF fibroblast signature. Multivariant analysis showed that DTF fibroblast signature was significantly more prognostic than the proliferation status in ovarian carcinomas.
Our results suggest that the DTF fibroblast signature is a common tumor stroma signature in different types of cancer including ovarian, lung and colon carcinomas. Our findings provide further insight into the DTF fibroblast stromal responses across different types of carcinomas and their potential as prognostic and therapeutic targets.
PMCID: PMC3778157  PMID: 23804424
12.  In vivo ossification of a scaffold combining β-tricalcium phosphate and platelet-rich plasma 
Tricalcium phosphate (TCP) and platelet-rich plasma (PRP) are commonly used in bone tissue engineering. The aim of the present study was to investigate a composite that combined TCP with PRP and assess its effectiveness in the treatment of bone defects. Cavity-shaped bone defects were established on the tibiae of 27 beagle dogs, and were repaired by pure β-TCP with bone marrow stromal cells (BMSCs), β-TCP/PRP with BMSCs and autogenic ilium. The samples were harvested at 4, 8 and 12 weeks, and bone regeneration was evaluated using X-ray radiography, immunocytochemical staining of osteocalcin (OCN), hematoxylin and eosin staining and reverse transcription-polymerase chain reaction analyses. Biomechanical tests of the scaffolds were performed at the 12th week after scaffold implantation. When using pure β-TCP as a scaffold, the scaffold-bone interface was clear and no material adsorption and bone healing was observed. Substantial bone regeneration was observed when the tibial defects were restored using β-TCP/PRP and autogenic ilium. Furthermore, the mRNA expression levels of OCN, alkaline phosphatase and collagen type I α1 were significantly higher in the animals with β-TCP/PRP scaffolds at 8 and 12 weeks following implantation compared with those in the animals with the pure β-TCP scaffolds. The maximum load and compressive strength of the β-TCP/PRP scaffolds were similar to those of the autogenic ilium; however, they were significantly higher than those of the pure β-TCP scaffold. Thus, the β-TCP/PRP composite may be used as a potential scaffold to carry in vitro cultured BMSCs to treat bone defects.
PMCID: PMC4186334  PMID: 25289027
tricalcium phosphate; platelet-rich plasma; tissue engineering; bone defect; bone marrow stromal cells
13.  Passive Smoking Exposure from Partners as a Risk Factor for ER+/PR+ Double Positive Breast Cancer in Never-Smoking Chinese Urban Women: A Hospital-Based Matched Case Control Study 
PLoS ONE  2014;9(5):e97498.
The relationship between passive smoking exposure (PSE) and breast cancer risk is of major interest.
To evaluate the relationship between PSE from partners and breast cancer risk stratified by hormone-receptor (HR) status in Chinese urban women population.
Hospital-based matched case control study.
Chinese urban breast cancer patients without current or previous active smoking history in China Medical University 1st Hospital, Liaoning Province, China between Jan 2009 and Nov 2009.
Each breast cancer patient was matched 1∶1 with healthy controls by gender and age (±2 years) from the same hospital.
The authors used unconditional logistic regression analyses to estimate odds ratio for women with PSE from partners and breast cancer risk.
312 pairs were included in the study. Women who endured PSE had significantly increased risk of breast cancer (adjusted OR: 1.46; 95% CI: 1.05–2.03; P = 0.027), comparing with unexposed women. Women who exposed to >5 cigarettes/day also had significant increased risk (adjusted OR: 1.99; 95% CI: 1.28–3.10; P = 0.002), as were women exposed to passive smoke for 16–25 years (adjusted OR: 1.87 95% CI: 1.22–2.86; P = 0.004), and those exposed to > 4 pack-years (adjusted OR: 1.71 95% CI: 1.17–2.50; P = 0.004). Similar trends were significant for estrogen receptor (ER)/progesterone receptor (PR) double positive subgroup(adjusted OR: 1.71; 2.20; 1.99; 1.92, respectively), but not for ER+/PR−, ER−/PR+, or ER−/PR− subgroups.
limitations of the hospital-based retrospective study, lack of information on entire lifetime PSE and low statistical power.
Our findings provide further evidence that PSE from partners contributes to increased risk of breast cancer, especially for ER/PR double positive breast cancer, in Chinese urban women.
PMCID: PMC4035255  PMID: 24866166
14.  Strong anisotropic lifetime orientation distributions of a two-level quantum emitter around a plasmonic nanorod 
Nanoscale Research Letters  2014;9(1):194.
Spontaneous emission lifetime orientation distributions of a two-level quantum emitter in metallic nanorod structures are theoretically investigated by the rigorous electromagnetic Green function method. It was found that spontaneous emission lifetime strongly depended on the transition dipole orientation and the position of the emitter. The anisotropic factor defined as the ratio between the maximum and minimum values of the lifetimes along different dipole orientations can reach up to 103. It is much larger than those in dielectric structures which are only several times usually. Our results show that the localized plasmonic resonance effect provides a new degree of freedom to effectively control spontaneous emission by the dipole orientation of the quantum emitters.
PMCID: PMC4006315  PMID: 24808801
Surface plasmons; Spontaneous emission; Lifetime distribution; Nanorod; 78.67.Qa; 73.20.Mf; 42.50.-p
15.  Acquisition of Tense Marking in English-Speaking Children with Cochlear Implants: A Longitudinal Study 
This study investigated the development of tense markers (e.g., past tense –ed) in children with cochlear implants (CIs) over a 3-year span. Nine children who received CIs before 30 months of age participated in this study at three, four, and five years postimplantation. Nine typical 3-, 4-, and 5-year- olds served as control groups. All children participated in a story-retell task. Percent correct of tense marking in the task was computed. Within the groups, percent correct of tense marking changed significantly in children with CIs and in typical children who had more hearing experience. Across the groups, children with CIs were significantly less accurate in tense marking than typical children at four and five years postimplantation. In addition, the performance of tense marking in children with CIs was correlated with their speech perception skills at earlier time points. Errors of tense marking tended to be omission rather than commission errors in typical children as well as in children with CIs. The findings suggested that despite the perceptual and processing constraints, children who received CIs may learn tense marking albeit with a delayed pattern.
PMCID: PMC3697805  PMID: 23288713
16.  Gene expression changes in adipose tissue with diet- and/or exercise-induced weight loss 
Adipose tissue plays a role in obesity-related cancers via increased production of inflammatory factors, steroid hormones, and altered adipokines. The impact of weight loss on adipose-tissue gene expression may provide insights into pathways linking obesity with cancer risk. We conducted an ancillary study within a randomized trial of diet, exercise, or combined diet+exercise vs. control among overweight/obese postmenopausal women. In 45 women, subcutaneous adipose-tissue biopsies were performed at baseline and after 6 months and changes in adipose-tissue gene expression were determined by microarray with an emphasis on pre-specified candidate pathways, as well as by unsupervised clustering of >37,000 transcripts (Illumina). Analyses were conducted first by randomization group, and then by degree of weight change at 6-months in all women combined. At 6 months, diet, exercise and diet+exercise participants lost a mean of 8.8 kg, 2.5 kg, and 7.9 kg (all p<0.05 vs. no change in controls). There was no significant change in candidate-gene expression by intervention group. In analysis by weight-change category, greater weight loss was associated a decrease in 17β-hydroxysteroid dehydrogenase-1 (HSD17B1, p-trend<0.01) and leptin (LEP, p-trend<0.01) expression, and marginally significant increased expression of estrogen receptor-1 (ESR1, p-trend=0.08) and insulin-like growth factor binding protein-3 (IGFBP3, p-trend=0.08). Unsupervised clustering revealed 83 transcripts with statistically significant changes. Multiple gene-expression changes correlated with changes in associated serum biomarkers. Weight-loss was associated with changes in adipose-tissue gene expression after 6 months, particularly in two pathways postulated to link obesity and cancer, i.e., steroid-hormone metabolism and IGF signaling.
PMCID: PMC3738189  PMID: 23341572
Adiposity; gene expression; obesity; weight-loss; exercise; diet; leptin; sex hormones; inflammation; adipokines; human; randomized-controlled trial
17.  Consequences, measurement, and evaluation of the costs associated with adverse drug reactions among hospitalized patients in China 
Adverse drug reactions (ADRs) are a leading cause of morbidity in developed countries and represent a substantial burden on health-care resources. Many countries spent 15% to 20% of their hospital budgets to treat drug complications. However, few studies have measured the pharmacoeconomic effects of ADRs on hospitalized patients in China. The study estimates the costs of ADRs as identified from the spontaneous voluntary reports completed from healthcare professionals. To do so, we calculate these costs, determine the sum of Medicare payments and their proportion of total healthcare spending, and evaluate the incidence of ADRs, characteristics of hospitalized ADR patients, and outcomes of ADRs in China.
This retrospective survey studied patients who experienced ADRs during their hospitalization at a Chinese tertiary-care teaching hospital. The patients were divided into group A and group B according to general ADRs and serious ADRs in Provisions for Adverse Drug Reaction Monitoring and Reporting. The direct costs included treatment fees, inspection fees, laboratory fees, materials fees, bed charges, drug charges, nursing care, meals, and other expenses and the sunk-cost losses were calculated according to the hospital information system (HIS). Indirect costs of ADR treatment were calculated according to the human capital approach. The epidemiological characteristics of ADRs were evaluated.
2739 were diagnosed with ADR during the study period, which translates to an ADR rate of 0.81%. The total socioeconomic loss from 2739 cases of ADR was estimated at ¥817401.69, consisting of direct costs of ¥603252.81 and indirect costs of ¥214148.88. On average, the costs per patient amounted to ¥196.10 in group A, ¥7032.29 in group B. The sum of medicare payment and proportion were ¥219061.13 (65.23%) and ¥105422.02 (39.42%) in group A and B. The ADR incidence in old-age patients was significantly higher than in other age groups (P < 0.0001). The most common drug class associated with ADRs represented antibiotics (957 patients, 34.94%).
The costs of especially severe ADRs could not be ignored, and in this hospital 0.13% of patients were diagnosed with ADRs associated with relatively higher direct costs than who suffered from mild ADRs, largely due to extended hospitalization.
PMCID: PMC3931293  PMID: 24533894
Adverse drug reaction; Direct medical cost; Hospitalized patients; Tertiary care teaching hospital; Pharmacoeconomic
18.  Consonant Development in Pediatric Cochlear Implant Users Who Were Implanted Before 30 Months of Age 
This study provided a yearly record of consonant development for the initial 4 years of cochlear implant (CI) use and established a precedent for using a standardized articulation test, the Goldman–Fristoe Test of Articulation—2 (Goldman, R., & Fristoe, M. [2000]. Goldman–Fristoe Test of Articulation—2. Circle Pines, MN: American Guidance Services). The study used CI age as a referent for 32 children who received their CI before 30 months of age. Consonants produced by 70% of the children were listed, as were the most common error types, which were consonant omissions and substitutions. Using consonant repertoire lists and standard scores, the study revealed that children with CIs had acquisition patterns that were similar to their peers when the duration of CI experience was similar to the chronological age norms of typically developing children. The results revealed that CI users need time to coordinate their articulatory organizing principles with the input they receive from their CI. It is appropriate to use length of CI use as a proxy for chronological age during the first 4 years when comparing articulation development with hearing peers.
PMCID: PMC3521776  PMID: 23143855
19.  Acidosis induces reprogramming of cellular metabolism to mitigate oxidative stress 
Cancer & Metabolism  2013;1:23.
A variety of oncogenic and environmental factors alter tumor metabolism to serve the distinct cellular biosynthetic and bioenergetic needs present during oncogenesis. Extracellular acidosis is a common microenvironmental stress in solid tumors, but little is known about its metabolic influence, particularly when present in the absence of hypoxia. In order to characterize the extent of tumor cell metabolic adaptations to acidosis, we employed stable isotope tracers to examine how acidosis impacts glucose, glutamine, and palmitate metabolism in breast cancer cells exposed to extracellular acidosis.
Acidosis increased both glutaminolysis and fatty acid β-oxidation, which contribute metabolic intermediates to drive the tricarboxylic acid cycle (TCA cycle) and ATP generation. Acidosis also led to a decoupling of glutaminolysis and novel glutathione (GSH) synthesis by repressing GCLC/GCLM expression. We further found that acidosis redirects glucose away from lactate production and towards the oxidative branch of the pentose phosphate pathway (PPP). These changes all serve to increase nicotinamide adenine dinucleotide phosphate (NADPH) production and counter the increase in reactive oxygen species (ROS) present under acidosis. The reduced novel GSH synthesis under acidosis may explain the increased demand for NADPH to recycle existing pools of GSH. Interestingly, acidosis also disconnected novel ribose synthesis from the oxidative PPP, seemingly to reroute PPP metabolites to the TCA cycle. Finally, we found that acidosis activates p53, which contributes to both the enhanced PPP and increased glutaminolysis, at least in part, through the induction of G6PD and GLS2 genes.
Acidosis alters the cellular metabolism of several major metabolites, which induces a significant degree of metabolic inflexibility. Cells exposed to acidosis largely rely upon mitochondrial metabolism for energy generation to the extent that metabolic intermediates are redirected away from several other critical metabolic processes, including ribose and glutathione synthesis. These alterations lead to both a decrease in cellular proliferation and increased sensitivity to ROS. Collectively, these data reveal a role for p53 in cellular metabolic reprogramming under acidosis, in order to permit increased bioenergetic capacity and ROS neutralization. Understanding the metabolic adaptations that cancer cells make under acidosis may present opportunities to generate anti-tumor therapeutic agents that are more tumor-specific.
PMCID: PMC4178214  PMID: 24359630
20.  Characterizing and quantifying leukocyte populations in human adipose tissue: impact of enzymatic tissue processing 
Journal of immunological methods  2012;386(1-2):50-59.
Adipose tissue inflammation is a major mechanistic link between obesity and chronic disease. To isolate and characterize specific leukocyte populations, e.g. by flow cytometry, tissue needs to be processed to digest the extracellular matrix. We have systematically compared the impact of different commonly used collagenase preparations, digestion times, and normalization strategies on the reproducibility of flow cytometric phenotyping of adipose tissue leukocyte populations. Subcutaneous adipose tissue was obtained from 11 anonymous donors undergoing elective procedures at a plastic surgery clinic in Seattle, WA. We found that collagenase alone consistently produced better cell yields (p=0.007) than when combined with additional proteases such as the commercially available liberases. Moreover, liberase significantly degraded the cell surface expression of CD4 (p<0.001) on T cells and to a lesser extent CD16 (p=0.058) on neutrophils. Extension of the digestion interval from 30 to 120 min did not significantly impact cell viability (p=0.319) or yield (p=0.247). Normalization by either ‘live-gate’, or percentage of CD45pos leukocytes exhibited the lowest coefficient of variation for tissue digests between 60 and 75 min, compared to normalization per gram of tissue, which consistently exhibited the greatest variability. Our data suggest that digestion of adipose tissue using pure collagenase for 60 to 75 min provides the best cell yield and viability, with minimal degradation of cell surface markers used to identify immune cell subpopulations, and best reproducibility independent of the normalization strategy.
PMCID: PMC3478958  PMID: 22974837
adipose tissue; leukocytes; enzymatic digestion; normalization
21.  Involvement of the Serine Protease Inhibitor, SERPINE2, and the Urokinase Plasminogen Activator in Cumulus Expansion and Oocyte Maturation 
PLoS ONE  2013;8(8):e74602.
The serpin peptidase inhibitor, clade E, member 2 (SERPINE2) inhibits urokinase-type plasminogen activator (PLAU) and tissue-type plasminogen activator. Higher SERPINE2 expression levels were detected in cumulus cells of human immature oocytes than in those of mature oocytes. The objective of this study was to evaluate whether high SERPINE2 levels in cumulus cells are associated with oocyte immaturity. Using the mouse cumulus–oocyte complex as an experimental model, the effects of elimination and overexpression of SERPINE2 in cumulus cells on cumulus expansion and oocyte maturation were assayed by in vitro maturation. Serpine2 and PLAU transcripts were the most highly expressed serpins and plasminogen activators, respectively. Their expression was coordinately regulated in cumulus cells during gonadotropin-induced oocyte maturation. Silencing of Serpine2 expression using small interfering RNAs or blockage of SERPINE2 protein using a specific antibody had no effect on oocyte maturation. However, overexpression of Serpine2 or exogenous supplementation with high levels of SERPINE2 impaired cumulus expansion and oocyte maturation, probably by decreasing hyaluronan synthase 2 (Has2) and versican (Vcan) mRNA expression. Amiloride, a specific PLAU inhibitor, also suppressed these processes. PLAU supplementation of the oocyte in vitro maturation medium caused earlier and more extensive expansion of cumulus cells and oocyte maturation that may be mediated by increased Has2 mRNA expression. However, these effects were neutralized by coincubation with SERPINE2 or amiloride and PLAU. In conclusion, SERPINE2 and PLAU are involved in cumulus expansion and oocyte maturation. High SERPINE2 levels impair these processes, probably by decreasing cumulus matrix gene expression as well as reducing cumulus hyaluronan contents and inhibiting PLAU activity. These findings may explain why cumulus cells surrounding immature human oocytes express high SERPINE2 levels.
PMCID: PMC3758271  PMID: 24023701
22.  Nicotine Promotes Mammary Tumor Migration via A Signaling Cascade Involving PKC and cdc42 
Cancer research  2008;68(20):8473-8481.
Nicotine, one of major components in tobacco, has been shown to be at high concentrations in the blood stream of cigarette smokers. However, the mechanisms of how nicotine affects tumor development and whether nicotine is a potential carcinogen for malignancies induced by second-hand smoking are not fully understood yet. Here, we investigate the signaling pathways by which nicotine potentiates tumorigenesis in human mammary epithelial-like MCF10A or cancerous MCF7 cells. We demonstrate that human MCF10A and MCF7 cells both express four subunits of nAChR. The treatment of these cells with nicotine enhances the activity of protein kinase C (PKC) α without altering the expression level of this kinase. Nicotine also stimulates [3H]thymidine incorporation into the genome of these cells as well as forces serum-starved cells to enter S phase of the cell cycle, resulting of growth promotion. Importantly, upon nicotine treatment, the mobility of MCF10A and MCF7 cells are enhanced, which can be blocked by the addition of nAChR or PKC inhibitor. Experiments using siRNA knockdown or ectopic expression of cdc42 demonstrated that cdc42 functions as a downstream effector of PKC and is crucial in the regulation of nicotine-mediated migratory activity in the cells. Together, our findings suggest that nicotine, through interacting with its receptor, initiates a signaling cascade that involves PKC and cdc42, and consequently promotes migration in mammary epithelial or tumor cells.
PMCID: PMC3698481  PMID: 18922921
23.  A novel small peptide as an epidermal growth factor receptor targeting ligand for nanodelivery in vitro 
The epidermal growth factor receptor (EGFR) serves an important function in the proliferation of tumors in humans and is an effective target for the treatment of cancer. In this paper, we studied the targeting characteristics of small peptides (AEYLR, EYINQ, and PDYQQD) that were derived from three major autophosphorylation sites of the EGFR C-terminus domain in vitro. These small peptides were labeled with fluorescein isothiocyanate (FITC) and used the peptide LARLLT as a positive control, which bound to putative EGFR selected from a virtual peptide library by computer-aided design, and the independent peptide RALEL as a negative control. Analyses with flow cytometry and an internalization assay using NCI-H1299 and K562 with high EGFR and no EGFR expression, respectively, indicated that FITC-AEYLR had high EGFR targeting activity. Biotin-AEYLR that was specifically bound to human EGFR proteins demonstrated a high affinity for human non-small-cell lung tumors. We found that AEYLR peptide-conjugated, nanostructured lipid carriers enhanced specific cellular uptake in vitro during a process that was apparently mediated by tumor cells with high-expression EGFR. Analysis of the MTT assay indicated that the AEYLR peptide did not significantly stimulate or inhibit the growth activity of the cells. These findings suggest that, when mediated by EGFR, AEYLR may be a potentially safe and efficient delivery ligand for targeted chemotherapy, radiotherapy, and gene therapy.
PMCID: PMC3632632  PMID: 23626467
EGFR; small peptide; tumor targeting; lung cancer; NLC
24.  Dual control of Shuanghuang Shengbai granule on upstream and downstream signal modulators of CyclinD-CDK4/6 signaling pathway of cell cycle in Lewis-bearing mice with cyclophosphamide-induced myelosuppression 
OncoTargets and therapy  2013;6:199-209.
This study investigated the dual control mechanism of the Shuanghuang Shengbai granule in modulating the cell cycle in Lewis-bearing mice with cyclophosphamide induced myelosuppression.
Thirty Lewis-bearing mice were randomly grouped into an untreated group, control group, and treated group. Both treated and untreated groups were intraperitoneally injected with cyclophosphamide to produce a myelosuppression model. Mice in the treated group were fed with the Shuanghuang Shengbai granule (40 g/day) for 6 consecutive days. Standard blood tests and the count of bone marrow nuclear cells were performed, and the cell reproductive cycles of bone marrow and tumors were measured in these mice. In addition, the western blot approach was used to measure the upstream activating signals of CyclinD-CDK4/6 such as c-Myc and CDC25A, the upstream suppression signals such as p16INK4a, and the expression of downstream activated signals such as Rb, pRB, and E2F. All of the tested results were validated by reverse transcription quantitative real-time polymerase chain reaction.
The results showed that the Shuanghuang Shengbai granule could elevate the count of leukocyte and bone marrow nuclear cells of Lewis-bearing mice with cyclophosphamide induced myelosuppression. It could also stimulate bone marrow cells to move from G0/G1 phases to S phase, accelerating the progress of the cell reproductive cycle and increasing the cell proliferation index. Simultaneously, the Shuanghuang Shengbai granule could also suppress cancer cells moving from G0/G1 phase to S phase, reducing the proliferation index. The tumor weight of Lewis-bearing mice in the treated group was much less than those of the control group. Expression levels of c-Myc, CDC25A, Rb, pRb, and E2F of bone marrow in Shuanghuang Shengbai granule-treated mice was higher compared to the control group, whereas they were lower in the cancer cells.
The experimental results demonstrate that the Shuanghuang Shengbai granule has dual control on the cell reproductive cycles in cancer cells and bone marrow nuclear cells in Lewis-bearing mice.
PMCID: PMC3615883  PMID: 23569384
Shuanghuang Shengbai granule; CyclinD-CDK4/6; c-Myc; Rb; pRb; E2F
25.  Prone breast forward intensity-modulated radiotherapy for Asian women with early left breast cancer: factors for cardiac sparing and clinical outcomes 
Journal of Radiation Research  2013;54(5):899-908.
Since December 2009, after breast-conserving surgery for Stage 0–I cancer of the left breast, 21 women with relatively pendulous breasts underwent computed tomography prone and supine simulations. The adjuvant radiotherapy was 50 Gy in 25 fractions to the left breast alone. Four plans—conventional wedged tangents and forward intensity-modulated radiotherapy (fIMRT) in supine and prone positions—were generated. fIMRT generated better homogeneity in both positions. Prone position centralized the breast tissue by gravity and also shortened the breast width which led to better conformity in both planning techniques. Prone fIMRT significantly reduced doses to left lung, Level I and Level II axilla. The mean cardiac doses did not differ between positions. Among the four plans, prone fIMRT produced the best target dosimetry and normal organ sparing. In subgroup analysis, patients with absolute breast depth > 7 cm in the prone position or breast depth difference > 3 cm between positions had significant cardiac sparing with prone fIMRT. Sixteen patients with significant cardiac sparing in prone position were treated using prone fIMRT and the others using supine fIMRT. All patients received a supine electron tumor bed boost of 10 Gy in 5 fractions. No patients developed Grade 2 or worse acute or late toxicities. There was no difference in the number of segments or beams, monitor units, treatment time, or positioning reproducibility between prone and supine positions. At a median follow-up time of 26.8 months, no locoregional or distant recurrence or death was noted.
PMCID: PMC3766291  PMID: 23504450
breast cancer; prone breast radiotherapy; intensity-modulated radiotherapy; breast tangents; Asian women

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