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1.  Upregulation of TLRs and IL-6 as a Marker in Human Colorectal Cancer 
Toll-like receptors (TLRs) not only form an important part of the innate immune system but also serve to activate the adaptive immune system in response to cancer. Real-time PCR; immunohistochemical stain and Western blotting analyses were performed to clarify molecular alterations in colorectal cancer (CRC) patients. We identified Toll-like receptor 1 (TLR1), TLR2, TLR4 and TLR8 gene expression levels and downstream gene, i.e., interleukin-6 (IL-6), IL-8, interferon-α (IFN-α) and myeloid differentiation primary-response protein-88 (MyD88), expression levels in CRC patients and in cancer cell lines. CRC tissues have higher TLR1, TLR2, TLR4, TLR8, IL-6 and IL-8 gene expression levels than do the normal colon mucosa (p < 0.05). TLR2 expression varied in different cell types (mucosa and lymphocytes). There was no difference in the MyD88 and IFN-α gene expression levels between cancerous and normal colon mucosa. CRC patients had higher levels of IL-6 (p = 0.002) and IL-8 (p = 0.038) expression than healthy volunteers did; and higher IL-6 and IL-8 expression was also found to signify a higher risk of recurrence. CL075 (3M002) treatments can reduce the production of IL-8 in different cancer cell lines. The signaling pathway of TLRs in cancer tissue is different from that in normal cells; and is MyD88-independent. Higher expression levels of TLR1, TLR2, TLR 4 and TLR 8 mRNA were related to upregulation inflammatory cytokines IL-6 and IL-8 gene expression in tissue and to the upregulation of IL-6 in blood. The concentration of IL-6 in serum can be used as an indicator of the possibility of CRC recurrence. Treatment with 3M002 can reduce IL-6 production in vitro and may prevent CRC recurrence. Our findings provide evidence that TLR1, TLR2, TLR4 and TLR8 gene expression induce downstream IL-6 and IL-8 gene expression; detection of these expression levels can serve as a CRC marker.
doi:10.3390/ijms16010159
PMCID: PMC4307241  PMID: 25547486
Toll-like receptors; real-time PCR; immunohistochemical stain; Western blotting; colorectal cancer
2.  Whole genome expression profiling of normal human fetal and adult ocular tissues 
Experimental eye research  2013;116:10.1016/j.exer.2013.08.009.
To study growth and development of ocular tissues, gene expression patterns in normal human fetal versus adult eyes were compared. Human retina/retinal pigment epithelium, choroid, sclera, optic nerve* and cornea* tissues were dissected from fetal (24 week gestational age) (N = 9; *N = 6), and adult (N = 6) normal donor eyes. The Illumina® whole genome expression microarray platform was used to assess differential expression. Statistical significance for all comparisons was determined using the Benjamin and Hochberg False Discovery Rate (FDR, 5%). Significant gene expression fold changes > 1.5 were found in adult versus fetal retina/RPE (N = 1185), choroid (N = 6446), sclera (N = 1349), and cornea (N = 3872), but not optic nerve. Genes showing differential expression were assessed using Ingenuity Pathway Analysis (IPA) for enriched functions and canonical pathways. In all tissues, development, cell death/growth, cancer functions, and signaling canonical pathways were enriched. There was also a general trend of down-regulation of collagen genes in adult tissues.
doi:10.1016/j.exer.2013.08.009
PMCID: PMC3875233  PMID: 24016867
gene expression; microarray; retina; retinal pigment epithelium; choroid; sclera; optic nerve; cornea
3.  Amifostine alleviates radiation-induced lethal small bowel damage via promotion of 14-3-3σ-mediated nuclear p53 accumulation 
Oncotarget  2014;5(20):9756-9769.
Amifostine (AM) is a radioprotector that scavenges free radicals and is used in patients undergoing radiotherapy. p53 has long been implicated in cell cycle arrest for cellular repair after radiation exposure. We therefore investigated the protective p53-dependent mechanism of AM on small bowel damage after lethal whole-abdominal irradiation (WAI). AM increased both the survival rate of rats and crypt survival following lethal 18 Gy WAI. The p53 inhibitor PFT-α compromised AM-mediated effects when administered prior to AM administration. AM significantly increased clonogenic survival in IEC-6 cells expressing wild type p53 but not in p53 knockdown cells. AM significantly increased p53 nuclear accumulation and p53 tetramer expression before irradiation through the inhibition of p53 degradation. AM inhibited p53 interactions with MDM2 but enhanced p53 interactions with 14-3-3σ. Knockdown of 14-3-3σ also compromised the effect of AM on clonogenic survival and p53 nuclear accumulation in IEC-6 cells. For the first time, our data reveal that AM alleviates lethal small bowel damage through the induction of 14-3-3σ and subsequent accumulation of p53. Enhancement of the p53/14-3-3σ interaction results in p53 tetramerization in the nucleus that rescues lethal small bowel damage.
PMCID: PMC4259435  PMID: 25230151
amifostine; p53; small bowel; whole-abdominal irradiation; 14-3-3σ; MDM2
4.  Subchondral Bone Trabecular Integrity Predicts and Changes Concurrently with Radiographic and MRI Determined Knee Osteoarthritis Progression 
Arthritis and rheumatism  2013;65(7):1812-1821.
Objective
To evaluate subchondral bone trabecular integrity (BTI) from a radiograph as a predictor of knee osteoarthritis (OA) progression.
Methods
Longitudinal (baseline, 12- and 24-month) knee radiographs were available from 60 female subjects with knee OA. OA progression was defined by 12- and 24-month change in radiographic medial compartment minimal joint space width (JSW) and medial joint space area (JSA), and medial tibial and femoral cartilage volume from magnetic resonance imaging. Bone Trabecular Integrity (BTI) of the medial tibial plateau was analyzed by fractal signature analysis with a commercially available software. Receiver Operating Characteristic curves of BTI were used to predict 5% change in OA progression parameters.
Results
Individual terms (linear and quadratic) of baseline BTI of vertical trabeculae predicted knee OA progression based on 12- and 24-month change in JSA (p<0.01 for 24 months), 24-month change in tibial (p<0.05) but not femoral cartilage volume, and 24-month change in JSW (p=0.05). ROC utilizing both terms of baseline BTI predicted 5% change in the OA progression parameters over 24 months with high accuracy as reflected by the area under the curve (AUC) measures: JSW 81%, JSA 85%, tibial 75% and femoral 85% cartilage volume. Change in BTI was also significantly associated (p<0.05) with concurrent change in JSA over 12 and 24 months and change in tibial cartilage volume over 24 months.
Conclusions
BTI predicts structural OA progression as determined by radiographic and MRI outcomes. BTI may therefore be worthy of study as an outcome measure for OA studies and clinical trials.
doi:10.1002/art.37970
PMCID: PMC4152231  PMID: 23576116
5.  The Alaris auditory evoked potential monitor as an indicator of seizure inducibility and duration during electroconvulsive therapy: an observational study 
BMC Anesthesiology  2014;14:34.
Background
Precise control of anesthetic depth during electroconvulsive therapy (ECT) is crucial because most intravenous anesthetics have anticonvulsant effects. In this study, we investigated the association between anesthetic depth measured by the Alaris auditory evoked potential index (AAI) and seizure inducibility and seizure duration during ECT.
Methods
Sixty-four ECTs were evaluated in 12 consecutive patients. General anesthesia was performed with a thiopental-based method. The relationship between the pre-ictal AAI, seizure activity and seizure duration was analyzed, and a possible threshold pre-ictal AAI to induce a seizure duration of at least 25 seconds was calculated.
Results
Forty-one of the 64 ECT stimuli successfully induced seizure activity that lasted longer than 25 seconds. Pre-ictal AAI was significantly correlated to seizure duration (r = 0.54, p < 0.001) and the threshold pre-ictal AAi value was calculated to be 26 (area under curve: 0.76, sensitivity: 70.3% and specificity: 73.9%, p < 0.001). ECT with a pre-ictal AAI ≧ 26 had a higher incidence of successful seizure activity ( p < 0.001) and a longer seizure duration (55 ± 35 v.s. 21 ± 27 seconds, p < 0.001).
Conclusion
Maintenance of a pre-ictal AAI value ≧ 26 was associated with an increased incidence of successful seizure activities and a longer seizure duration. This is the first report to investigate Alaris AEP monitoring during ECT.
doi:10.1186/1471-2253-14-34
PMCID: PMC4049489  PMID: 24914401
Auditory evoked potential; Electroconvulsive therapy; Seizure inducibility
6.  Effect of Age-Related Cartilage Turnover on Serum C-Telopeptide of Collagen Type II and Osteocalcin Levels in Growing Rabbits with and without Surgically Induced Osteoarthritis 
BioMed Research International  2014;2014:284784.
This study aims to determine the effect of age-related cartilage turnover on the serum C-telopeptide of type II collagen (CTX-II) and osteocalcin (OC) levels in growing rabbits with and without surgically induced osteoarthritis. Twenty-four New Zealand male 3-month-old rabbits were randomized into three operated groups (n = 6 per group, with surgically induced osteroarthritis in the right knee; after blood sampling, the knees were harvested following euthanization at 2, 3, and 6 months after surgery) and a control group (n = 6, blood samples were obtained monthly between 3 and 15 months). Histomorphologically, the medial femoral condyles, particularly the central parts, harbored the most severe osteoarthritic changes among the operated rabbits. The serum levels of CTX-II and OC decreased in the controls from 3 to 11 months and then remained stable. No significant differences in the serum CTX-II and OC levels between the osteoarthritic rabbits and controls were observed. The osteoarthritic-to-normal ratios (ONRs, the ratios of serum CTX-II or OC levels in osteoarthritic rabbits to those of the controls at same ages) enabled an overall assessment of osteoarthritis and age-related cartilage turnover. Elevated CTX-II ONRs were observed in rabbits with mild to advanced osteoarthritis. However, the OC ONRs were unhelpful in assessing osteoarthritic growing rabbits.
doi:10.1155/2014/284784
PMCID: PMC3963374  PMID: 24729965
7.  Progress in the Prevention of Mother to Child Transmission of HIV in Three Regions of Tanzania: A Retrospective Analysis 
PLoS ONE  2014;9(2):e88679.
Background
Mother to child transmission (MTCT) of HIV-1 remains an important problem in sub-Saharan Africa where most new pediatric HIV-1 infections occur. Early infant diagnosis of HIV-1 using dried blood spot (DBS) PCR among exposed infants provides an opportunity to assess current MTCT rates.
Methods
We conducted a retrospective data analysis on mother-infant pairs from all PMTCT programs in three regions of northern Tanzania to determine MTCT rates from 2008–2010. Records of 3,016 mother-infant pairs were assessed to determine early transmission among HIV-exposed infants in the first 75 days of life.
Results
Of 2,266 evaluable infants in our cohort, 143 had a positive DBS PCR result at ≤75 days of life, for an overall transmission rate of 6.3%. Transmission decreased substantially over the period of study as more effective regimens became available. Transmission rates were tightly correlated to maternal regimen: 14.9% (9.5, 20.3) of infants became infected when women received no therapy; 8.8% (6.9, 10.7) and 3.6% (2.4, 4.8) became infected when women received single-dose nevirapine (sdNVP) or combination prophylaxis, respectively; the lowest MTCT rates occurred when women were on HAART, with 2.1% transmission (0.3, 3.9). Treatment regimens changed dramatically over the study period, with an increase in combination prophylaxis and a decrease in the use of sdNVP. Uptake of DBS PCR more than tripled over the period of study for the three regions surveyed.
Conclusions
Our study demonstrates significant reductions in MTCT of HIV-1 in three regions of Tanzania coincident with increased use of more effective PMTCT interventions. The changes we demonstrate for the period of 2008–2010 occurred prior to major changes in WHO PMTCT guidelines.
doi:10.1371/journal.pone.0088679
PMCID: PMC3923804  PMID: 24551134
8.  Association Mapping of the High-Grade Myopia MYP3 Locus Reveals Novel Candidates UHRF1BP1L, PTPRR, and PPFIA2 
Purpose.
Myopia, or nearsightedness, is a common ocular genetic disease for which over 20 candidate genomic loci have been identified. The high-grade myopia locus, MYP3, has been reported on chromosome 12q21–23 by four independent linkage studies.
Methods.
We performed a genetic association study of the MYP3 locus in a family-based high-grade myopia cohort (n = 82) by genotyping 768 single-nucleotide polymorphisms (SNPs) within the linkage region. Qualitative testing for high-grade myopia (sphere ≤ −5 D affected, > −0.5 D unaffected) and quantitative testing on the average dioptric sphere were performed.
Results.
Several genetic markers were nominally significantly associated with high-grade myopia in qualitative testing, including rs3803036, a missense mutation in PTPRR (P = 9.1 × 10−4) and rs4764971, an intronic SNP in UHRF1BP1L (P = 6.1 × 10−4). Quantitative testing determined statistically significant SNPs rs4764971, also found by qualitative testing (P = 3.1 × 10−6); rs7134216, in the 3′ untranslated region (UTR) of DEPDC4 (P = 5.4 × 10−7); and rs17306116, an intronic SNP within PPFIA2 (P < 9 × 10−4). Independently conducted whole genome expression array analyses identified protein tyrosine phosphatase genes PTPRR and PPFIA2, which are in the same gene family, as differentially expressed in normal rapidly growing fetal relative to normal adult ocular tissue (confirmed by RT-qPCR).
Conclusions.
In an independent high-grade myopia cohort, an intronic SNP in UHRF1BP1L, rs4764971, was validated for quantitative association, and SNPs within PTPRR (quantitative) and PPFIA2 (qualitative and quantitative) approached significance. Three genes identified by our association study and supported by ocular expression and/or replication, UHRF1BP1L, PTPRR, and PPFIA2, are novel candidates for myopic development within the MYP3 locus that should be further studied.
Association mapping of the MYP3 locus reveals novel candidate genes.
doi:10.1167/iovs.12-11102
PMCID: PMC3621505  PMID: 23422819
9.  Trabecular Morphometry by Fractal Signal Analysis is a Novel Marker of Osteoarthritis Progression 
Arthritis and rheumatism  2009;60(12):3711-3722.
Objective
To evaluate the utility of subchondral bone texture from a baseline x-ray image for predicting 3-year knee osteoarthritis (OA) progression.
Methods
A total of 138 participants in the Prediction of Osteoarthritis Progression (POP) study were evaluated at baseline and 3 years. Fixed-flexion knee radiographs of the 248 non-replaced knees underwent fractal analysis of the medial subchondral tibial plateau using a commercially available software tool. OA progression was defined as a 1-grade change in joint space narrowing (JSN) or osteophyte based on a standardized knee atlas. Statistical analysis of fractal signatures was performed using a new method based on modeling the overall shape of fractal dimension versus radius curves.
Results
Baseline fractal signature of the medial tibial plateau was predictive of medial knee JSN progression (area under the curve [AUC] of Receiver Operating Characteristic plot of 0.75), but not progression based on osteophyte or progression of the lateral compartment. The traditional covariates (age, gender, body mass index, knee pain), general bone mineral content, and baseline joint space width fared little better than random variables for predicting OA progression (AUC 0.52–0.58). The maximal predictive model combined baseline fractal signature, knee alignment, traditional covariates, and bone mineral content (AUC 0.79).
Conclusions
We identified a prognostic marker of OA that is readily extracted from a plain radiograph by fractal signature analysis. The global shape approach to analyzing these data is a potentially efficient means of identifying individuals at risk of knee OA progression that needs to be validated in a second cohort.
doi:10.1002/art.25012
PMCID: PMC3711179  PMID: 19950282
osteoarthritis; imaging; biomarker; subchondral bone
10.  Whole Body Bone Scintigraphy Provides a Measure of Total Body Burden of Osteoarthritis for the Purpose of Systemic Biomarker Validation 
Arthritis and rheumatism  2009;60(11):3366-3373.
Objective
To evaluate the association of serum and synovial fluid cartilage oligomeric matrix protein (COMP) with systemic and local measures of osteoarthritis (OA) activity by bone scintigraphy.
Methods
Knee joint synovial fluid (total 275 knees) and serum were obtained from 159 participants with symptomatic OA of at least one knee. Technetium-99m-methylene diphosphonate (Tc-MDP) bone scintigraphy was performed and early phase knee scans and late phase whole body bone scans of 15 additional joint sites were scored semi-quantitatively. Correlations of bone scan scores with COMP utilized generalized linear modelling to control for within subject correlation of knee data. Principal components analysis was used to explore the contribution of each joint site to the variance in serum COMP.
Results
The correlation of synovial fluid and serum COMP was significant (r=0.206, p=0.006). Synovial fluid COMP correlated most strongly with early phase knee bone scan scores (p=0.0003), even after adjustment for OA severity by late phase bone scan (p=0.015), as well as with synovial fluid volume (p<0.0001). Serum COMP correlated with total body bone scan scores (r=0.188, p=0.018) and with a factor composed of bone scan scores of the shoulders, spine, lateral knees and sacroiliac joints (p=0.0004).
Conclusion
Synovial fluid COMP correlated strongly with two indicators of knee joint inflammation: early phase bone scintigraphy and synovial fluid volume. Serum COMP correlated with total body joint disease severity by late phase bone scintigraphy, supporting the hypothesis that whole body bone scintigraphy is a means of quantifying total body burden of OA for systemic biomarker validation.
doi:10.1002/art.24856
PMCID: PMC3692562  PMID: 19877068
osteoarthritis; biomarkers; bone scintigraphy; principal components analysis; COMP
11.  Association of Bone Scintigraphic Abnormalities with Knee Malalignment and Pain 
Annals of the rheumatic diseases  2008;68(11):1673-1679.
Objective
We evaluated the information content of knee bone scintigraphy, including pattern, localization and intensity of retention relative to radiographic features of knee osteoarthritis (rOA), knee alignment, and knee symptoms.
Methods
A total of 308 knees (159 subjects) with symptomatic and radiographic knee OA (rOA) of at least one knee were assessed by late phase technetium-99m-methylene disphosphonate bone scintigraph, fixed-flexion knee radiograph, full limb radiograph for knee alignment, and for self-reported knee symptom severity. Generalized linear models were used to control for within subject correlation of knee data.
Results
The compartmental localization (medial versus lateral) and intensity of knee bone scan retention were associated with the pattern (varus versus valgus) (p<0.001) and severity (p=0.0008) of knee malalignment, and localization and severity of rOA (p<0.0001). Bone scan agent retention in the tibiofemoral, but not patellofemoral compartment, was associated with severity of knee symptoms (p=0.0009), and persisted after adjusting for rOA (p=0.0012).
Conclusion
To our knowledge, this is the first study describing a relationship between knee malalignment, joint symptom severity, and compartment specific abnormalities by bone scintigraphy. This work demonstrates that bone scintigraphy as a sensitive and quantitative indicator of symptomatic knee OA. Used selectively, bone scintigraphy is a dynamic imaging modality that holds great promise as a clinical trial screening tool and outcome measure.
doi:10.1136/ard.2008.094722
PMCID: PMC3684623  PMID: 18981032
osteoarthritis; bone scintigraphy; malalignment; knee
12.  Ecological Facilitation between Two Epiphytes through Drought Mitigation in a Subtropical Rainforest 
PLoS ONE  2013;8(5):e64599.
Positive species interactions (facilitation) play an important role in shaping the structures and species diversity of ecological communities, particularly under stressful environmental conditions. Epiphytes in rainforests often grow in multiple-species clumps, suggesting interspecies facilitation. However, little is known about the patterns and mechanisms of epiphyte co-occurrence. We assessed the interactions of two widespread epiphyte species, Asplenium antiquum and Haplopteris zosterifolia, by examining their co-occurrence and size-class association in the field. To elucidate factors controlling their interactions, we conducted reciprocal-removal and greenhouse-drought experiments, and nutrient and isotope analyses. Forty-five percent of H. zosterifolia co-occurred with A. antiquum, whereas only 17% of A. antiquum co-occurred with H. zosterifolia. Removing the fronds plus substrate of A. antiquum reduced the relative frond length and specific leaf area of H. zosterifolia, but removing fronds only had little effect. Removing H. zosterifolia had no significant effects on the growth of A. antiquum. H. zosterifolia co-occurring and not co-occurring with A. antiquum had similar foliar nutrient concentrations and δ15N values, suggesting that A. antiquum does not affect the nutrient status of H. zosterifolia. Reduced growth of H. zosterifolia with the removal of A. antiquum substrate, together with higher foliar δ13C for H. zosterifolia growing alone than those co-occurring with A. antiquum, suggest that A. antiquum enhances water availability to H. zosterifolia. This enhancement probably resulted from water storage in the substrate of A. antiquum, which could hold water up to 6.2 times its dry weight, and from reduced evapotranspiration due to shading of A. antiquum fronds. Greater water loss occurred in the frond-clipped group than the unclipped group between days 3–13 of the drought treatment. Our results imply that drought mitigation by substrate-forming epiphytes is important for maintaining epiphyte diversity in tropic and subtropic regions with episodic water limitations, especially in the context of anthropogenic climate change.
doi:10.1371/journal.pone.0064599
PMCID: PMC3669308  PMID: 23741346
13.  Slow Freezing Coupled Static Magnetic Field Exposure Enhances Cryopreservative Efficiency—A Study on Human Erythrocytes 
PLoS ONE  2013;8(3):e58988.
The aim of this study was to assess the cryoprotective effect of static magnetic fields (SMFs) on human erythrocytes during the slow cooling procedure. Human erythrocytes suspended in 20% glycerol were slowly frozen with a 0.4-T or 0.8-T SMF and then moved to a −80°C freezer for 24 hr. The changes in survival rate, morphology, and metabolites of the thawed erythrocytes were examined. To understand possible cryoprotective mechanisms of SMF, membrane fluidity and dehydration stability of SMF-exposed erythrocytes were tested. For each test, sham-exposed erythrocytes were used as controls. Our results showed that freezing coupled with 0.4-T or 0.8-T SMFs significantly increased the relative survival ratios of the frozen-thawed erythrocytes by 10% and 20% (p<0.001), respectively. The SMFs had no effect on erythrocyte morphology and metabolite levels. However, membrane fluidity of the samples exposed to 0.8-T SMF decreased significantly (p<0.05) in the hydrophobic regions. For the dehydration stability experiments, the samples exposed to 0.8-T SMF exhibited significantly lower (p<0.05) hemolysis. These results demonstrate that a 0.8-T SMF decreases membrane fluidity and enhances erythrocyte membrane stability to resist dehydration damage caused by slow cooling procedures.
doi:10.1371/journal.pone.0058988
PMCID: PMC3592815  PMID: 23520546
14.  Extracorporeal shockwave therapy shows a number of treatment related chondroprotective effect in osteoarthritis of the knee in rats 
Background
Extracorporeal shockwave therapy (ESWT) shows chondroprotective effect in osteoarthritis of the rat knees. However, the ideal number of ESWT is unknown. This study investigated the effects of different numbers of ESWT in osteoarthritis of the knee in rats.
Methods
Forty-five male Sprague-Dawley rats were divided into five groups. Group I underwent sham arthrotomy without anterior cruciate ligament transection (ACLT) or medial meniscectomy (MM) and received no ESWT. Group II underwent ACLT + MM and received no ESWT. Group III underwent ACLT + MM, and received ESWT once a week for one treatment. Group IV underwent ACLT + MM and received ESWT twice a week for 2 treatments. Group V underwent ACLT + MM and received ESWT three times a week for 3 treatments. Each treatment consisted of 800 impulses of shockwave at 14 Kv to the medial tibia condyle. The evaluations included radiographs of the knee, histomorphological examination and immunohistochemical analysis at 12 weeks.
Results
At 12 weeks, group II and V showed more radiographic arthritis than groups I, III and IV. On histomorphological examination, the Safranin O matrix staining in groups III and IV are significantly better than in groups II and V, and the Mankin scores in groups III and IV are less than groups II and V. Groups III and IV showed significant decreases of Mankin score and increase of Safranin O stain as compared to group I. Group V showed significant increases of Mankin score and a decrease of Safranin O stain as compared to group II. In articular cartilage, group II showed significant increase of MMP13 and decrease of collagen II as compared to group I. Groups III and IV showed significant decrease of MMP13 and increase of collagen II as compared to group I. Group V showed significant increase of MMP13 and decrease of collagen II as compared to group II. In subchondral bone, vWF, VEGF, BMP-2 and osteocalcin significantly decreased in groups II and V, but increased in groups III and IV relative to group I.
Conclusions
ESWT shows a number of treatment related chondroproctective effect in osteoarthritis of the knee in rats.
doi:10.1186/1471-2474-14-44
PMCID: PMC3626641  PMID: 23356403
Shockwave; Number of treatment; Chondroprotective; Osteoarthritis; Knee; Rats
15.  A phase I study on pharmacokinetics and pharmacodynamics of higenamine in healthy Chinese subjects 
Acta Pharmacologica Sinica  2012;33(11):1353-1358.
Aim:
To investigate the pharmacokinetics, pharmacodynamics, and safety of higenamine, an active ingredient of Aconite root, in healthy Chinese volunteers.
Methods:
Ten subjects received continuous, intravenous infusion of higenamine at gradually escalating doses from 0.5 to 4.0 μg·kg−1·min−1, each dose was given for 3 min. Blood and urine samples were collected at designated time points to measure the concentrations of higenamine. Pharmacodynamics was assessed by measuring the subject's heart rate. A nonlinear mixed-effect modeling approach, using the software Phoenix NLME, was used to model the plasma concentration-time profiles and heart rate.
Results:
Peak concentrations (Cmax) of higenamine ranged from 15.1 to 44.0 ng/mL. The half-life of higenamine was 0.133 h (range, 0.107–0.166 h), while the area under concentration-time curve (AUC), extrapolated to infinity, was 5.39 ng·h·mL−1 (range, 3.2-6.8 ng·h·mL−1). The volume of distribution (V) was 48 L (range, 30.8–80.6 L). The total clearance (CL) was 249 L/h (range, 199-336 L/h). Within 8 h, 9.3% (range, 4.6%–12.4%) of higenamine was recovered in the urine. The pharmacokinetics of higenamine was successfully described using a two-compartment model with nonlinear clearance. In the pharmacodynamic model, heart rates were related to the plasma drug concentrations using a simple direct effect model with baseline. The E0, Emax, and EC50 were 68 bpm, 73 bpm and 8.1 μg/L, respectively.
Conclusion:
Higenamine has desirable pharmacokinetic and pharmacodynamic characteristics. The results provide important information for future clinical studies on higenamine.
doi:10.1038/aps.2012.114
PMCID: PMC4011356  PMID: 23085737
higenamine; Aconite root; coronary artery disease; stress myocardial perfusion imaging; pharmacologic stress test; pharmacokinetics; pharmacodynamics
16.  A single NFκB system for both canonical and non-canonical signaling 
Cell Research  2010;21(1):86-102.
Two distinct nuclear factor κB (NFκB) signaling pathways have been described; the canonical pathway that mediates inflammatory responses, and the non-canonical pathway that is involved in immune cell differentiation and maturation and secondary lymphoid organogenesis. The former is dependent on the IκB kinase adaptor molecule NEMO, the latter is independent of it. Here, we review the molecular mechanisms of regulation in each signaling axis and attempt to relate the apparent regulatory logic to the physiological function. Further, we review the recent evidence for extensive cross-regulation between these two signaling axes and summarize them in a wiring diagram. These observations suggest that NEMO-dependent and -independent signaling should be viewed within the context of a single NFκB signaling system, which mediates signaling from both inflammatory and organogenic stimuli in an integrated manner. As in other regulatory biological systems, a systems approach including mathematical models that include quantitative and kinetic information will be necessary to characterize the network properties that mediate physiological function, and that may break down to cause or contribute to pathology.
doi:10.1038/cr.2010.161
PMCID: PMC3193412  PMID: 21102550
NFκB; inflammation; immune response; immune development; mathematical model; signaling crosstalk
17.  An international collaborative family-based whole genome quantitative trait linkage scan for myopic refractive error 
Molecular Vision  2012;18:720-729.
Purpose
To investigate quantitative trait loci linked to refractive error, we performed a genome-wide quantitative trait linkage analysis using single nucleotide polymorphism markers and family data from five international sites.
Methods
Genomic DNA samples from 254 families were genotyped by the Center for Inherited Disease Research using the Illumina Linkage Panel IVb. Quantitative trait linkage analysis was performed on 225 Caucasian families and 4,656 markers after accounting for linkage disequilibrium and quality control exclusions. Two refractive quantitative phenotypes, sphere (SPH) and spherical equivalent (SE), were analyzed. The SOLAR program was used to estimate identity by descent probabilities and to conduct two-point and multipoint quantitative trait linkage analyses.
Results
We found 29 markers and 11 linkage regions reaching peak two-point and multipoint logarithms of the odds (LODs)>1.5. Four linkage regions revealed at least one LOD score greater than 2: chromosome 6q13–6q16.1 (LOD=1.96 for SPH, 2.18 for SE), chromosome 5q35.1–35.2 (LOD=2.05 for SPH, 1.80 for SE), chromosome 7q11.23–7q21.2 (LOD=1.19 for SPH, 2.03 for SE), and chromosome 3q29 (LOD=1.07 for SPH, 2.05 for SE). Among these, the chromosome 6 and chromosome 5 regions showed the most consistent results between SPH and SEM. Four linkage regions with multipoint scores above 1.5 are near or within the known myopia (MYP) loci of MYP3, MYP12, MYP14, and MYP16. Overall, we observed consistent linkage signals across the SPH and SEM phenotypes, although scores were generally higher for the SEM phenotype.
Conclusions
Our quantitative trait linkage analyses of a large myopia family cohort provided additional evidence for several known MYP loci, and identified two additional potential loci at chromosome 6q13–16.1 and chromosome 5q35.1–35.2 for myopia. These results will benefit the efforts toward determining genes for myopic refractive error.
PMCID: PMC3324362  PMID: 22509102
19.  Copy Number Variation of KIR Genes Influences HIV-1 Control 
PLoS Biology  2011;9(11):e1001208.
The authors that the number of activating and inhibitory KIR genes varies between individuals and plays a role in the regulation of immune mechanisms that determine HIV-1 control.
A genome-wide screen for large structural variants showed that a copy number variant (CNV) in the region encoding killer cell immunoglobulin-like receptors (KIR) associates with HIV-1 control as measured by plasma viral load at set point in individuals of European ancestry. This CNV encompasses the KIR3DL1-KIR3DS1 locus, encoding receptors that interact with specific HLA-Bw4 molecules to regulate the activation of lymphocyte subsets including natural killer (NK) cells. We quantified the number of copies of KIR3DS1 and KIR3DL1 in a large HIV-1 positive cohort, and showed that an increase in KIR3DS1 count associates with a lower viral set point if its putative ligand is present (p = 0.00028), as does an increase in KIR3DL1 count in the presence of KIR3DS1 and appropriate ligands for both receptors (p = 0.0015). We further provide functional data that demonstrate that NK cells from individuals with multiple copies of KIR3DL1, in the presence of KIR3DS1 and the appropriate ligands, inhibit HIV-1 replication more robustly, and associated with a significant expansion in the frequency of KIR3DS1+, but not KIR3DL1+, NK cells in their peripheral blood. Our results suggest that the relative amounts of these activating and inhibitory KIR play a role in regulating the peripheral expansion of highly antiviral KIR3DS1+ NK cells, which may determine differences in HIV-1 control following infection.
Author Summary
There is marked intrinsic variation in the extent to which individuals are able to control HIV-1. We have identified a genetic copy number variable region (CNV) in humans that plays a significant role in the control of HIV-1. This CNV is located in the genomic region that encodes the killer cell immunoglobulin-like receptors (KIRs) and specifically affects the KIR3DS1 and KIR3DL1 genes, encoding two KIRs that interact with human leukocyte antigen B (HLA-B) ligands. KIRs are expressed on the surface of natural killer (NK) cells, which serve as important players in the innate immune response, and are involved in the recognition of infected and malignant cells through a loss or alteration in “self” ligands. We use both genetic association and functional evidence to show a strong interaction between KIR3DL1 and KIR3DS1, indicating that increasing gene counts for KIR3DL1 confer increasing levels of protection against HIV-1, but only in the presence of at least one copy of KIR3DS1. This effect was associated with a dramatic increase in the abundance of KIR3DS1+ NK cells in the peripheral blood, and strongly associated with a more robust capacity of peripheral NK cells to suppress HIV-1 replication in vitro. This work provides one of the few examples of an association between a relatively common CNV and a human complex trait.
doi:10.1371/journal.pbio.1001208
PMCID: PMC3226550  PMID: 22140359
20.  Multi-objective optimization of enzyme manipulations in metabolic networks considering resilience effects 
BMC Systems Biology  2011;5:145.
Background
Improving the synthesis rate of desired metabolites in metabolic systems is one of the main tasks in metabolic engineering. In the last decade, metabolic engineering approaches based on the mathematical optimization have been used extensively for the analysis and manipulation of metabolic networks. Experimental evidence shows that mutants reflect resilience phenomena against gene alterations. Although researchers have published many studies on the design of metabolic systems based on kinetic models and optimization strategies, almost no studies discuss the multi-objective optimization problem for enzyme manipulations in metabolic networks considering resilience phenomenon.
Results
This study proposes a generalized fuzzy multi-objective optimization approach to formulate the enzyme intervention problem for metabolic networks considering resilience phenomena and cell viability. This approach is a general framework that can be applied to any metabolic networks to investigate the influence of resilience phenomena on gene intervention strategies and maximum target synthesis rates. This study evaluates the performance of the proposed approach by applying it to two metabolic systems: S. cerevisiae and E. coli. Results show that the maximum synthesis rates of target products by genetic interventions are always over-estimated in metabolic networks that do not consider the resilience effects.
Conclusions
Considering the resilience phenomena in metabolic networks can improve the predictions of gene intervention and maximum synthesis rates in metabolic engineering. The proposed generalized fuzzy multi-objective optimization approach has the potential to be a good and practical framework in the design of metabolic networks.
doi:10.1186/1752-0509-5-145
PMCID: PMC3203348  PMID: 21929795
21.  Modulation of Notch-1 Signaling Alleviates Vascular Endothelial Growth Factor–Mediated Diabetic Nephropathy 
Diabetes  2010;59(8):1915-1925.
OBJECTIVE
Disturbances in podocytes are typically associated with marked proteinuria, a hallmark of diabetic nephropathy. This study was conducted to investigate modulation of Notch-1 signaling in high glucose (HG)-stressed human podocytes and in a diabetic animal model.
RESEARCH DESIGN AND METHODS
Expression of the Notch signaling components was examined in HG-treated podocytes, human embryonic kidney cells (HEK293), and kidneys from diabetic animals by RT-qPCR, Western blot analysis, and immunohistochemical staining. The association between the Notch signaling, VEGF expression, and podocyte integrity was evaluated.
RESULTS
Notch-1 signaling was significantly activated in HG-cultured human podocytes and HEK293 cells and kidneys from diabetic animals. HG also augmented VEGF expression, decreasing nephrin expression and podocyte number—a critical event for the development of proteinuria in diabetic nephropathy. After use of pharmacological modulators or specific shRNA knockdown strategies, inhibition of Notch-1 signaling significantly abrogated VEGF activation and nephrin repression in HG-stressed cells and ameliorated proteinuria in the diabetic kidney.
CONCLUSIONS
Our findings suggest that upregulation of Notch-1 signaling in HG-treated renal podocytes induces VEGF expression and subsequent nephrin repression and apoptosis. Modulation of Notch-1 signaling may hold promise as a novel therapeutic strategy for the treatment of diabetic nephropathy.
doi:10.2337/db09-0663
PMCID: PMC2911050  PMID: 20522599
22.  A unified sample preparation protocol for proteomic and genomic profiling of cervical swabs to identify biomarkers for cervical cancer screening 
Proteomics. Clinical applications  2008;2(12):1658-1669.
Cervical cancer screening is ideally suited for the development of biomarkers due to the ease of tissue acquisition and the well-established histological transitions. Furthermore, cell and biologic fluid obtained from cervix samples undergo specific molecular changes that can be profiled. However, the ideal manner and techniques for preparing cervical samples remains to be determined. To address this critical issue a patient screening protein and nucleic acid collection protocol was established. RNAlater was used to collect the samples followed by proteomic methods to identify proteins that were differentially expressed in normal cervical epithelial versus cervical cancer cells. Three hundred ninety spots were identified via two-dimensional difference gel electrophoresis (2-D DIGE) that were expressed at either higher or lower levels (>3-fold) in cervical cancer samples. These proteomic results were compared to genes in a cDNA microarray analysis of microdissected neoplastic cervical specimens to identify overlapping patterns of expression. The most frequent pathways represented by the combined dataset were: cell cycle: G2/M DNA damage checkpoint regulation; aryl hydrocarbon receptor signaling; p53 signaling; cell cycle: G1/S checkpoint regulation; and the endoplasmic reticulum stress pathway. HNRPA2B1 was identified as a biomarker candidate with increased expression in cancer compared to normal cervix and validated by Western blot.
doi:10.1002/prca.200780146
PMCID: PMC3042129  PMID: 21136816
2-D DIGE; biomarkers; cervical cancer; cDNA microarray; RNAlater
23.  GWAPower: a statistical power calculation software for genome-wide association studies with quantitative traits 
BMC Genetics  2011;12:12.
Background
In designing genome-wide association (GWA) studies it is important to calculate statistical power. General statistical power calculation procedures for quantitative measures often require information concerning summary statistics of distributions such as mean and variance. However, with genetic studies, the effect size of quantitative traits is traditionally expressed as heritability, a quantity defined as the amount of phenotypic variation in the population that can be ascribed to the genetic variants among individuals. Heritability is hard to transform into summary statistics. Therefore, general power calculation procedures cannot be used directly in GWA studies. The development of appropriate statistical methods and a user-friendly software package to address this problem would be welcomed.
Results
This paper presents GWAPower, a statistical software package of power calculation designed for GWA studies with quantitative traits, where genetic effect is defined as heritability. Based on several popular one-degree-of-freedom genetic models, this method avoids the need to specify the non-centrality parameter of the F-distribution under the alternative hypothesis. Therefore, it can use heritability information directly without approximation. In GWAPower, the power calculation can be easily adjusted for adding covariates and linkage disequilibrium information. An example is provided to illustrate GWAPower, followed by discussions.
Conclusions
GWAPower is a user-friendly free software package for calculating statistical power based on heritability in GWA studies with quantitative traits. The software is freely available at: http://dl.dropbox.com/u/10502931/GWAPower.zip
doi:10.1186/1471-2156-12-12
PMCID: PMC3036643  PMID: 21255436
24.  Bis[4-(2-isopropyl-2H-tetra­zol-5-yl)phen­yl]dimethyl­silane 
The title compound, C22H28N8Si, has crystallographic 2 symmetry with the Si atom located on a twofold rotation axis. The tetra­zole ring is oriented at a dihedral angle of 5.32 (18)° with respect to the benzene ring. A C—H⋯π inter­action occurs between adjacent mol­ecules in the crystal structure.
doi:10.1107/S160053681100033X
PMCID: PMC3051474  PMID: 21523046
25.  The tricarboxylic acid cycle in Shewanella oneidensis is independent of Fur and RyhB control 
BMC Microbiology  2010;10:264.
Background
It is well established in E. coli and Vibrio cholerae that strains harboring mutations in the ferric uptake regulator gene (fur) are unable to utilize tricarboxylic acid (TCA) compounds, due to the down-regulation of key TCA cycle enzymes, such as AcnA and SdhABCD. This down-regulation is mediated by a Fur-regulated small regulatory RNA named RyhB. It is unclear in the γ-proteobacterium S. oneidensis whether TCA is also regulated by Fur and RyhB.
Results
In the present study, we showed that a fur deletion mutant of S. oneidensis could utilize TCA compounds. Consistently, expression of the TCA cycle genes acnA and sdhA was not down-regulated in the mutant. To explore this observation further, we identified a ryhB gene in Shewanella species and experimentally demonstrated the gene expression. Further experiments suggested that RyhB was up-regulated in fur mutant, but that AcnA and SdhA were not controlled by RyhB.
Conclusions
These cumulative results delineate an important difference of the Fur-RyhB regulatory cycle between S. oneidensis and other γ-proteobacteria. This work represents a step forward for understanding the unique regulation in S. oneidensis.
doi:10.1186/1471-2180-10-264
PMCID: PMC2964681  PMID: 20950482

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