Reconstitution of tumor development in immunodeficient mice from disaggregated primary human tumor cells is always challenging. The main goal of the present study is to establish a reliable assay system that would allow us to reproducibly reconstitute human prostate tumor regeneration in mice using patient tumor-derived single cells. Using many of the 114 untreated primary human prostate cancer (HPCa) samples we have worked on, here we show that: 1) the subcutaneum represents the most sensitive site that allows the grafting of the implanted HPCa pieces; 2) primary HPCa cells by themselves fail to regenerate tumors in immunodeficient hosts; 3) when coinjected in Matrigel with rUGM (rat urogenital sinus mesenchyme), CAF (carcinoma-associated fibroblasts), or Hs5 (immortalized bone marrow derived stromal) cells, primary HPCa cells fail to initiate serially transplantable tumors in NOD/SCID mice; and 4) however, HPCa cells coinjected with the Hs5 cells into more immunodeficient NOD/SCID-IL2Rγ−/− (NSG) mice readily regenerate serially transplantable tumors. The HPCa/Hs5 reconstituted ‘prostate’ tumors present an overall epithelial morphology, are of the human origin, and contain cells positive for AR, CK8, and racemase. Cytogenetic analysis provides further evidence for the presence of karyotypically abnormal HPCa cells in the HPCa/Hs5 tumors. Of importance, HPCa/Hs5 xenograft tumors contain EpCAM+ cells that are both clonogenic and tumorigenic. Surprisingly, all HPCa/Hs5 reconstituted tumors are undifferentiated, even for HPCa cells derived from Gleason 7 tumors. Our results indicate that primary HPCa cells coinjected with the immortalized Hs5 stromal cells generate undifferentiated tumors in NSG mice and we provide evidence that undifferentiated HPCa cells might be the cells that possessed tumorigenic potential and regenerated HPCa/Hs5 xenograft tumors.
Inflammatory mechanisms are important in stroke risk, and genetic variations in components of the inflammatory response have been implicated as risk factors for stroke. We tested the inflammatory gene polymorphisms and their association with ischemic stroke in a Chinese Han population.
A total of 1,124 ischemic stroke cases and 1,163 controls were genotyped with inflammatory panel strips containing 51 selected inflammatory gene polymorphisms from 35 candidate genes. We tested the genotype-stroke association with logistic regression model.
We found two single nucleotide polymorphisms (SNPs) in CCL11 were associated with ischemic stroke. After adjusting for multiple testing using false discovery rate (FDR) with a 0.20 cut-off point, CCL11 rs4795895 remained statistically significant. We further stratified the study population by their hypertension status. In the hypertensive group, CCR2 rs1799864, CCR5 rs1799987 and CCL11 rs4795895 were nominally associated with increased risk of stroke. In the non-hypertensive group, CCL11 rs3744508, LTC4S rs730012, FCER1B rs569108, TGFB1 rs1800469, LTA rs909253 and CCL11 rs4795895 were associated with ischemic stroke. After correction for multiple testing, CCR2 rs1799864 and CCR5 rs1799987 remained significant in the hypertensive group, and CCL11 rs3744508, LTC4S rs730012, FCER1B rs569108, TGFB1 rs1800469, LTA rs909253 remained significant in the non-hypertensive group.
Our results indicate that inflammatory genetic variants are associated with increased risk of ischemic stroke in a Chinese Han population, particularly in non-hypertensive individuals.
Association study; Hypertension; Inflammatory gene; Ischemic stroke
Cancer cell molecular mimicry of stem cells (SC) imbues neoplastic cells with enhanced proliferative and renewal capacities. In support, numerous mediators of SC self-renewal have been evinced to exhibit oncogenic potential. We have recently reported that shRNA-mediated knockdown of the embryonic stem cell (ESC) self-renewal gene NANOG significantly reduced the clonogenic and tumorigenic capabilities of various cancer cells. In this study, we sought to test the potential pro-tumorigenic functions of NANOG, particularly, in prostate cancer (PCa). Using quantitative RT-PCR, we first confirmed that PCa cells expressed NANOG mRNA primarily from the NANOGP8 locus on chromosome 15q14. We then constructed a lentiviral promoter reporter in which the -3.8 kb NANOGP8 genomic fragment was used to drive the expression of green fluorescence protein (GFP). We observed that NANOGP8-GFP+ PCa cells exhibited cancer stem cell (CSC) characteristics such as enhanced clonal growth and tumor regenerative capacity. To further investigate the functions and mechanisms of NANOG in tumorigenesis, we established tetracycline-inducible NANOG overexpressing cancer cell lines, including both prostate (Du145 and LNCaP) and breast (MCF-7) cancer cells. NANOG induction promoted drug-resistance in MCF-7 cells, tumor regeneration in Du145 cells, and, most importantly, castration-resistant tumor development in LNCaP cells. These pro-tumorigenic effects of NANOG were associated with key molecular changes, including an upregulation of molecules such as CXCR4, IGFBP5, CD133 and ALDH1. The present gain-of-function studies, coupled with our recent loss-of-function work, establish the integral role for NANOG in neoplastic processes and shed light on its mechanisms of action.
Nanog; prostate cancer; cancer stem cells; castration resistance; self-renewal
This study investigated the associations of plasma leptin levels with insulin resistance (IR) and prediabetes in relatively lean, rural Chinese men and women.
Design and methods
This study included 574 subjects aged 21–45 years from a community-based twin cohort. Plasma leptin concentrations were measured by sandwich immunoassays using flowemetric xMAP technology. Prediabetes was defined based on fasting plasma glucose and 75g oral glucose tolerance test. Multivariate linear and logistic regression analyses were used to investigate gender-specific associations of leptin with IR measures and prediabetes, adjusting for intra-twin correlation, measures of adiposity, and other pertinent covariates.
The body mass index(BMI) is 22.3±2.7 kg/m2 in men and 22.5±2.7 kg/m2 in women. Leptin levels were positively associated with IR. Individuals with higher tertiles of leptin also had increased risk of prediabetes with OR of 2.6 (95%CI: 1.4–5.1) and 4.3 (95%CI: 2.1–8.7) in men; OR of 1.1 (95%CI: 0.6–2.1) and 3.1 (95%CI 1.5–6.2) in women for 2nd and 3rd tertile, respectively. These associations were attenuated after further adjusting for adiposity measurements only in men. The Leptin-prediabetes associations disappeared after adjusting for the homeostatic model assessment of insulin resistance (HOMA-IR) in both genders.
In this sample of relatively lean rural Chinese adults, plasma leptin levels were associated with IR and prediabetes in a dose-response fashion, which were not totally explained by adiposity. Our data underscored that prediabetes is not all about obesity, and leptin may be an additional biomarker for screening individuals at high risk for prediabetes in this population.
Leptin; insulin resistance; prediabetes; Chinese
Chronic pain is a major health concern that affects millions of people. There are no adequate long-term therapies for chronic pain sufferers, leading to significant cost for both society and the individual. The most commonly used therapy for chronic pain is the application of opioid analgesics and nonsteroidal anti-inflammatory drugs, but these drugs can lead to addiction and may cause side effects. Further studies of the mechanisms of chronic pain have opened the way for development of new treatment strategies, one of which is gene therapy. The key to gene therapy is selecting safe and highly efficient gene delivery systems that can deliver therapeutic genes to overexpress or suppress relevant targets in specific cell types. Here we review several promising viral vectors that could be applied in gene transfer for the treatment of chronic pain and further discuss the possible mechanisms of genes of interest that could be delivered with viral vectors for the treatment of chronic pain.
Mutations in GJB2 are the most common molecular defects responsible for autosomal recessive nonsyndromic hearing impairment (NSHI). The mutation spectra of this gene vary among different ethnic groups.
In order to understand the spectrum and frequency of GJB2 mutations in the Chinese population, the coding region of the GJB2 gene from 2063 unrelated patients with NSHI was PCR amplified and sequenced.
A total of 23 pathogenic mutations were identified. Among them, five (p.W3X, c.99delT, c.155_c.158delTCTG, c.512_c.513insAACG, and p.Y152X) are novel. Three hundred and seven patients carry two confirmed pathogenic mutations, including 178 homozygotes and 129 compound heterozygotes. One hundred twenty five patients carry only one mutant allele. Thus, GJB2 mutations account for 17.9% of the mutant alleles in 2063 NSHI patients. Overall, 92.6% (684/739) of the pathogenic mutations are frame-shift truncation or nonsense mutations. The four prevalent mutations; c.235delC, c.299_c.300delAT, c.176_c.191del16, and c.35delG, account for 88.0% of all mutantalleles identified. The frequency of GJB2 mutations (alleles) varies from 4% to 30.4% among different regions of China. It also varies among different sub-ethnic groups.
In some regions of China, testing of the three most common mutations can identify at least one GJB2 mutant allele in all patients. In other regions such as Tibet, the three most common mutations account for only 16% the GJB2 mutant alleles. Thus, in this region, sequencing of GJB2 would be recommended. In addition, the etiology of more than 80% of the mutant alleles for NSHI in China remains to be identified. Analysis of other NSHI related genes will be necessary.
Neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF), are promising therapeutic agents for neurodegenerative diseases. However, the application of GDNF to treat these diseases effectively is limited because the blood–brain barrier (BBB) prevents the local delivery of macromolecular therapeutic agents from entering the central nervous system (CNS). Focused ultrasound combined with microbubbles (MBs) using appropriate parameters has been previously demonstrated to be able to open the BBB locally and noninvasively. This study investigated the targeted delivery of GDNF MBs through the BBB by magnetic resonance imaging (MRI)-guided focused ultrasound. Evans Blue extravasation and histological examination were used to determine the optimum focused ultrasound parameters. Enzyme-linked immunosorbent assay was performed to verify the effects of GDNF bound on MBs using a biotin–avidin bridging chemistry method to promote GDNF delivery into the brain. The results showed that GDNF can be delivered locally and noninvasively into the CNS through the BBB using MRI-guided focused ultrasound combined with MBs under optimum parameters. MBs that bind GDNF combined with MRI-guided focused ultrasound may be an effective way of delivering neurotrophic factors directly into the CNS. The method described herein provides a potential means of treating patients with CNS diseases.
Src plays various roles in tumour progression, invasion, metastasis, angiogenesis and survival. It is one of the multiple targets of multi-target kinase inhibitors in clinical uses and trials for the treatment of leukemia and other cancers. These successes and appearances of drug resistance in some patients have raised significant interest and efforts in discovering new Src inhibitors. Various in-silico methods have been used in some of these efforts. It is desirable to explore additional in-silico methods, particularly those capable of searching large compound libraries at high yields and reduced false-hit rates.
We evaluated support vector machines (SVM) as virtual screening tools for searching Src inhibitors from large compound libraries. SVM trained and tested by 1,703 inhibitors and 63,318 putative non-inhibitors correctly identified 93.53%~ 95.01% inhibitors and 99.81%~ 99.90% non-inhibitors in 5-fold cross validation studies. SVM trained by 1,703 inhibitors reported before 2011 and 63,318 putative non-inhibitors correctly identified 70.45% of the 44 inhibitors reported since 2011, and predicted as inhibitors 44,843 (0.33%) of 13.56M PubChem, 1,496 (0.89%) of 168 K MDDR, and 719 (7.73%) of 9,305 MDDR compounds similar to the known inhibitors.
SVM showed comparable yield and reduced false hit rates in searching large compound libraries compared to the similarity-based and other machine-learning VS methods developed from the same set of training compounds and molecular descriptors. We tested three virtual hits of the same novel scaffold from in-house chemical libraries not reported as Src inhibitor, one of which showed moderate activity. SVM may be potentially explored for searching Src inhibitors from large compound libraries at low false-hit rates.
Src; c-src; Computer aided drug design; Kinase inhibitor; Virtual screening; Support vector machine
It has been hypothesized that vitamin D deficiency (VDD) contributes to the development of food sensitization (FS) and then food allergy. However, the epidemiological evidence is conflicting. We aim to examine if cord blood VDD is associated with FS and if such association can be modified by genetic variants in a prospective birth cohort.
This study included 649 children who were enrolled at birth and followed from birth onward at the Boston Medical Center. We defined VDD as cord blood 25(OH)D < 11ng/ml, and FS as specific IgE ≥ 0.35kUA/L to any of eight common food allergens in early childhood. We genotyped potentially functional single nucleotide polymorphisms (SNPs) in 11 genes known to be involved in regulating IgE and 25(OH)D concentrations. Logistic regressions were used to test the effects of VDD on FS individually and jointly with SNPs.
Among the 649 children, 44% had VDD and 37% had FS. When examined alone, VDD was not associated with FS. When examined jointly with SNPs, a significant interaction between IL4 gene polymorphism (rs2243250) and VDD (pinteraction=0.003, pFDR=0.10) was found: VDD increased the risk of FS among children carrying CC/CT genotypes (OR=1.79, 95%CI: 1.15–2.77). Similar but weaker interactions were observed for SNPs in MS4A2 (rs512555), FCER1G (rs2070901), and CYP24A1 (rs2762934). When all four SNPs were simultaneously considered, a strong gene-VDD interaction was evident (pinteraction=9×10−6).
Our data demonstrate that VDD may increase the risk of FS among individuals with certain genotypes, providing evidence of gene-vitamin D interaction on FS.
cord blood plasma 25(OH)D; food sensitization; gene-vitamin D deficiency interaction; SNP
Spontaneous intracerebral hemorrhage is a disease with high morbidity, high disability rate, high mortality, and high economic burden. Whether patients can benefit from surgical evacuation of hematomas is still controversial, especially for those with moderate-volume hematomas in the basal ganglia. This study is designed to compare the efficacy of endoscopic surgery and conservative treatment for the moderate-volume hematoma in spontaneous basal ganglia hemorrhage.
Patients meet the criteria will be randomized into the endoscopic surgery group (endoscopic surgery for hematoma evacuation and the best medical treatment) or the conservative treatment group (the best medical treatment). Patients will be followed up at 1, 3, and 6 months after initial treatment. The primary outcomes include the Extended Glasgow Outcome Scale and the Modified Rankin Scale. The secondary outcomes consist of the National Institutes of Health Stroke Scale and the mortality. The Barthel Index(BI) will also be evaluated. The sample size is 100 patients.
The ECMOH trial is a randomized controlled trial designed to evaluate if endoscopic surgery is better than conservative treatment for patients with moderate-volume hematomas in the basal ganglia.
Chinese Clinical Trial Registry: ChiCTR-TRC-11001614
Endoscopic surgery; Conservative treatment; Moderate-volume hematoma; Spontaneous basal ganglia hemorrhage
DNA double-strand breaks (DSBs) are among the most lethal lesions associated with genome stability, which, when destabilized, predisposes organs to cancers. DSBs are primarily fixed either with little fidelity by non-homologous end joining (NHEJ) repair or with high fidelity by homology-directed repair (HDR). The phosphorylated form of H2AX on serine 139 (γ-H2AX) is a marker of DSBs. In this study, we explored if the protein phosphatase PP6 is involved in DSB repair by depletion of its expression in human cancer cell lines, and determined PP6 expression in human breast cancer tissues by immunohistochemistry staining. We found that bacterially produced PP6c (the catalytic subunit of PP6)-containing heterotrimeric combinations exhibit phosphatase activity against γ-H2AX in the in vitro phosphatase assays. Depletion of PP6c or PP6R2 led to persistent high levels of γ-H2AX after DNA damage and a defective HDR. Chromatin immunoprecipitation assays demonstrated that PP6c was recruited to the region adjacent to the DSB sites. Expression of PP6c, PP6R2 and PP6R3 in human breast tumors was significantly lower than those in benign breast diseases. Taken together, our results suggest that γ-H2AX is a physiological substrate of PP6 and PP6 is required for HDR and its expression may harbor a protective role during the development of breast cancer.
protein phosphatase; PP6; γ-H2AX; DNA double-strand break; homology-directed repair
It is important to develop novel antipsychotics that can effectively treat schizophrenia with minor side-effects. The aim of our work is to develop novel antipsychotics that act on dopamine D2 and D3, serotonin 5-HT1A and 5-HT2A receptors with low affinity for the serotonin 5-HT2C and H1 receptors, which can effectively cure positive symptoms, negative symptoms and cognitive impairment without the weight gain side-effect.
A series of 2-substituted-5-thiopropylpiperazine (piperidine) -1,3,4-oxadiazoles derivatives have been synthesized and the target compounds were evaluated for binding affinities to D2, 5-HT1A and 5-HT2A receptors. Preliminary results indicated that compounds 14, 16 and 22 exhibited high affinities to D2, 5-HT1A and 5-HT2A receptors among these compounds. Further binding tests showed that compound 22 had high affinity for D3 receptor, and low affinity for serotonin 5-HT2C and H1 receptors. In addition, compound 22 inhibited apomorphine-induced climbing behavior and MK-801-induced hyperactivity with no extrapyramidal symptoms liability in mice. Moreover, compound 22 exhibited acceptable pharmacokinetic properties.
Compound 22 showed an atypical antipsychotic activity without liability for extrapyramidal symptoms. We anticipate compound 22 to be useful for developing a novel class of drug for the treatment of schizophrenia.
In this work, we conducted functional analysis of Arabidopsis HRS1 gene in order to provide new insights into the mechanisms governing seed germination. Compared with wild type (WT) control, HRS1 knockout mutant (hrs1-1) exhibited significant germination delays on either normal medium or those supplemented with abscisic acid (ABA) or sodium chloride (NaCl), with the magnitude of the delay being substantially larger on the latter media. The hypersensitivity of hrs1-1 germination to ABA and NaCl required ABI3, ABI4 and ABI5, and was aggravated in the double mutant hrs1-1abi1-2 and triple mutant hrs1-1hab1-1abi1-2, indicating that HRS1 acts as a negative regulator of ABA signaling during seed germination. Consistent with this notion, HRS1 expression was found in the embryo axis, and was regulated both temporally and spatially, during seed germination. Further analysis showed that the delay of hrs1-1 germination under normal conditions was associated with reduction in the elongation of the cells located in the lower hypocotyl (LH) and transition zone (TZ) of embryo axis. Interestingly, the germination rate of hrs1-1 was more severely reduced by the inhibitor of cell elongation, and more significantly decreased by the suppressors of plasmalemma H+-ATPase activity, than that of WT control. The plasmalemma H+-ATPase activity in the germinating seeds of hrs1-1 was substantially lower than that exhibited by WT control, and fusicoccin, an activator of this pump, corrected the transient germination delay of hrs1-1. Together, our data suggest that HRS1 may be needed for suppressing ABA signaling in germinating embryo axis, which promotes the timely germination of Arabidopsis seeds probably by facilitating the proper function of plasmalemma H+-ATPase and the efficient elongation of LH and TZ cells.
Both long and short sleep duration have been associated with obesity, cardiovascular disease, and diabetes. However, there have been no previous studies investigating the potential relationship between altered sleep duration and allergen sensitization.
To explore the association between sleep duration and sensitization to food and aeroallergens.
This study includes 1534 rural Chinese adolescent twins aged 12 to 21 years who completed standard sleep questionnaires and skin prick tests (SPTs) to 9 food and 5 aeroallergens. Total sleep time was defined as the interval from bedtime to wake-up time minus sleep latency. Sensitization was defined as having at least one positive SPT.
Compared to individuals with the highest (3rd) tertile of sleep duration, those who slept less were more likely to be sensitized to any food allergen with odds ratios (ORs) of 1.9 (95% confidence interval(CI):1.3–2.7) and 1.4 (95%CI:1.0–1.9) for the 1st and 2nd tertiles (trend test Ptrend=3×10−4), respectively. The corresponding ORs for sensitization to any aeroallergen were 1.5 (95%CI: 1.1–2.0) and 1.3 (95%CI:1.0–1.7) (Ptrend=8×10−3). These associations were independent of percent body fat. In addition, we observed a significant dose-response association between the number of positive SPTs and percentage of shortest sleep duration (1st tertile) (Ptrend=1×10−3).
Conclusions and Clinical Relevance
In this sample of relatively lean rural Chinese adolescents, we found that short sleep duration was associated with increased risk of sensitization to food and aeroallergens, independent of percent body fat. Longitudinal studies are needed to further determine the temporal and causal relationships. If short sleep duration indeed is one of the risk factors for allergic sensitization, the global burden of allergic diseases could be dramatically reduced by providing appropriate guidance on sleep duration for youth.
sleep duration; skin prick test; allergen; sensitization; adolescent
Myxococcus fulvus HW-1 (ATCC BAA-855) is a halotolerant marine myxobacterium. This strain exhibits complex social behaviors in the presence of low concentrations of seawater but adopts an asocial living pattern under oceanic conditions. The whole genome of M. fulvus HW-1 will enable us to further investigate the details of its evolution.
Theranostic platform integrating diagnostic imaging and therapeutic function into a single system has become a new direction of nanoparticle research. In the process of treatment, therapeutic efficacy is monitored. The use of theranostic nanoparticle can add an additional "layer" to keep track on the therapeutic agent such as the pharmacokinetics and biodistribution. In this report, we have developed quantum rod (QR) based formulations for the delivery of small interfering RNAs (siRNAs) to human neuronal cells. PEGlyated QRs with different surface functional groups (amine and maleimide) were designed for selectively down-regulating the dopaminergic signaling pathway which is associated with the drug abuse behavior. We have demonstrated that the DARPP-32 siRNAs were successfully delivered to dopaminergic neuronal (DAN) cells which led to drastic knockdown of specific gene expression by both the electrostatic and covalent bond conjugation regimes. The PEGlyated surface offered high biocompatibilities and negligible cytotoxicities to the QR formulations that may facilitate the in vivo applications of these nanoparticles.
Quantum Rod; Gene Delivery; Addiction Gene Therapy; Phospholipid; PEG; siRNA.
Regulatory T lymphocytes (Treg) expressing the Forkhead Box Transcription Factor 3 (Foxp3) are critical modulators of autoimmunity. Foxp3+ Treg may develop in the thymus as a population distinct from conventional Foxp3− αβ T cells (Tconv). Alternatively, plasticity in Foxp3 expression may allow for the interconversion of mature Treg and Tconv. We examined >160,000 TCR sequences from Foxp3+ or Foxp3− populations in the spleens or CNS of wild type mice with experimental allergic encephalomyelitis (EAE) to determine their relatedness and identify distinguishing TCR features. Our results indicate that the CNS infiltrating Treg and Tconv arise predominantly from distinct sources. The repertoires of CNS Treg or Tconv TCR showed limited overlap with heterologous populations in either the CNS or spleen, indicating that they are largely unrelated. Indeed, Treg and Tconv TCR in the CNS were significantly less related than those populations in the spleen. In contrast, CNS Treg and Tconv repertoires strongly intersected those of the homologous cell type in the spleen. High frequency sequences more likely to be disease associated showed similar results, and some public TCR demonstrated Treg or Tconv-specific motifs. Different charge characteristics and amino acid use preferences were identified in the CDR3β of Treg and Tconv infiltrating the CNS, further indicating that their repertoires are qualitatively distinct. Therefore discrete populations of Treg and Tconv that do not substantially interconvert respond during EAE. Differences in sequence and physical characteristics distinguish Treg and Tconv TCR and imply dissimilar antigen recognition properties.
Knowledge and investigation of therapeutic targets (responsible for drug efficacy) and the targeted drugs facilitate target and drug discovery and validation. Therapeutic Target Database (TTD, http://bidd.nus.edu.sg/group/ttd/ttd.asp) has been developed to provide comprehensive information about efficacy targets and the corresponding approved, clinical trial and investigative drugs. Since its last update, major improvements and updates have been made to TTD. In addition to the significant increase of data content (from 1894 targets and 5028 drugs to 2025 targets and 17 816 drugs), we added target validation information (drug potency against target, effect against disease models and effect of target knockout, knockdown or genetic variations) for 932 targets, and 841 quantitative structure activity relationship models for active compounds of 228 chemical types against 121 targets. Moreover, we added the data from our previous drug studies including 3681 multi-target agents against 108 target pairs, 116 drug combinations with their synergistic, additive, antagonistic, potentiative or reductive mechanisms, 1427 natural product-derived approved, clinical trial and pre-clinical drugs and cross-links to the clinical trial information page in the ClinicalTrials.gov database for 770 clinical trial drugs. These updates are useful for facilitating target discovery and validation, drug lead discovery and optimization, and the development of multi-target drugs and drug combinations.
Mutations of cystic fibrosis (CF) transmembrane conductance regulator (CFTR) cause lethal hereditary disease CF that involves extensive destruction and dysfunction of serous epithelium. Possible pharmacological therapy includes correction of defective intracellular processing and abnormal channel gating. In a previous study, we identified five natural coumarin potentiators of ΔF508-CFTR including osthole, imperatorin, isopsoralen, praeruptorin A, and scoparone. The present study was designed to determine the activity of these coumarine compounds on CFTR activity in animal tissues as a primary evaluation of their therapeutic potential. In the present study, we analyzed the affinity of these coumarin potentiators in activating wild-type CFTR and found that they are all potent activators. Osthole showed the highest affinity with Kd values <50 nmol/L as determined by Ussing chamber short-circuit current assay. Stimulation of rat colonic mucosal secretion by osthole was tested by the Ussing chamber short-circuit current assay. Osthole reached maximal activation of colonic Cl− secretion at 5 μmol/L. Stimulation of mouse tracheal mucosal secretion was analyzed by optical measurement of single gland secretion. Fluid secretion rate of tracheal single submucosal gland stimulated by osthole at 10 μmol/L was three-fold more rapid than that in negative control. In both cases the stimulated secretions were fully abolished by CFTRinh-172. In conclusion, the effective stimulation of Cl– and fluid secretion in colonic and tracheal mucosa by osthole suggested the therapeutic potential of natural coumarin compounds for the treatment of CF and other CFTR-related diseases.
CFTR; coumarins; activator; fluid secretion; mucosa; colon; airway
Due to recent use of short-chain ceramides in preclinical studies, we characterized C6-ceramide metabolism in cancer cell lines and assessed metabolic junctures for enhancing efficacy. MDA-MB-231 breast cancer cells decreased the amount of C6-ceramide metabolized to C6-sphingomyelin (C6-SM) and increased the amount metabolized to C6-glucosylceramide (C6-GC) in response to increasing concentrations. A similar trend was seen in DU-145 (prostate cancer), PANC-1 (pancreatic cancer), and LoVo (colorectal cancer) cells. KG-1 leukemia cells favored C6-SM synthesis at low (0.6 μM) and high-dose (12 μM) C6-ceramide. Partnering C6-ceramide with tamoxifen, a P-glycoprotein antagonist that impedes ceramide glycosylation, was an effective regimen for enhancing cytotoxicity in cells. Experiments to assess the mechanism of cell death using KG-1 cells showed that tamoxifen inhibited synthesis of C6-GC and C6-SM from C6-ceramide by 80% and 50%, respectively, which was accompanied by enhanced apoptosis. Radiolabeling of KG-1 cells with [3H]palmitic acid produced a 2-fold increase in 3H-long-chain ceramides when unlabeled C6-ceramide was added and a 9-fold increase when C6-ceramide and tamoxifen were added. The increase in 3H-palmitate radiolabeling of long-chain ceramides was blocked by inclusion of a ceramide synthase inhibitor; however, inhibiting synthesis of long-chain ceramide did not rescue cells. These studies show that tamoxifen enhances the apoptotic effects of C6-ceramide. The proposed mechanism involves blocking short-chain ceramide anabolism to favor hydrolysis and generation of sphingosine. We propose that use of tamoxifen and other P-glycoprotein antagonists can be an effective means for enhancing cytotoxic potential of short-chain ceramides in the treatment of cancer.
ceramide; glucosylceramide; cancer cells; tamoxifen
This study was an attempt to examine the phenotypic, genetic, and environmental correlations between percent fat mass (PFM) and bone parameters, especially hip geometry, among 786 males and 618 females aged 13 to 21 years from a Chinese twin cohort. PFM, bone area (BA), bone mineral content (BMC), cross-sectional area (CSA), and section modulus (SM) were obtained by dual-energy X-ray absorptiometry. Multiple linear regression models were used to assess the PFM-bone relationships. A structural equation model for twin design was used to estimate genetic/environmental influences on individual phenotype and phenotypic correlations. After controlling for body weight and other pertinent covariates, we observed inverse associations between PFM and bone parameters: Compared with the lowest age- and gender-specific tertile of PFM, males in the highest tertile of PFM had lower measures of whole-body-less-head BA (WB-BA), lumbar spine BA (L2–L4-BA), total-hip BA (TH-BA), total-hip BMC, CSA, and SM (p < .005 for all, adjusted p < .05). Similar inverse associations were observed in females for all the preceding parameters except WB-BA and L2–L4-BA. These associations did not vary significantly by Tanner stages. In both genders, the estimated heritabilities were 80% to 86% for BMC, 67% to 80% for BA, 74% to 77% for CSA, and 64% for SM. Both shared genetics and environmental factors contributed to the inverse PFM-bone correlations. We conclude that in this sample of relatively lean Chinese adolescents, at a given body weight, PFM is inversely associated with BA, BMC, and hip geometry in both genders, and such associations are attributed to both shared genetic and environmental factors. © 2010 American Society for Bone and Mineral Research.
percent fat mass; hip geometry; bone mineral content; adolescence; coheritability
To estimate the effects of human umbilical vein (HUV) implanted under the sclera of glaucoma model on intraocular pressure (IOP) lowering and to investigate its related mechanisms
A total of 20 human umbilical veins (HUV) were collected from healthy fetus umbilical core. After the establishment of glaucoma model in rabbits, human freeze-dried umbilical vein was implanted under the sclera during NPDS, while for control group, sclerostomy was performed without implant. The formation of the filtration bleb and IOP were detected every 24 hours before surgery and on day 3, 7, 10 and 14 after surgery. Handheld pen-type Tono-pen II tonometer was used to measure IOP after topical anesthesia treatment. Each measurement has three duplicates. The incision recovery, filtration, conjunctiva congestion and anterior chamber inflammation were observed everyday after surgery.
IOP was decreased dramatically with less inflammation than traditional sclerostomies with the application of HUV. The significant differences of IOP between the NPDS with and without HUV implant groups were shown up from 10 days after surgery. The average IOP in NPDS without HUV implant was 14.25mmHg, while for NPDS with HUV implant group, it was 12.30mmHg. This structure of filtration bleb, which allowed the aqueous humor to leave the eye, was formed for any type of surgery. However, 1-2 weeks later, filtration bleb was still existed in the group of sclerostomy with HUV implant and more stable than that of the surgery without HUV implant. Histological observations were performed on day 3, 7 and 14 after surgery. For the eyes under sclerostomy with HUV implant, HUV lumina was shown up on 3 days after surgery with few fibroblast cells near the sclera. On 7 days after surgery, HUV lumina was stably maintained but with obvious fibroblast cells and inflammatory cell. On 14 days after surgery, HUV lumina was still clearly observed but with scarring formation, which suggests that the IOP lowering effects might result from an effective drainage structure formation.
HUV might be an alternative material to make the drainage pathway for non-penetrating deep sclerostomy.
IOP-lowering; non-penetrating deep sclerostomy; human umbilical vein
The source, specificity, and plasticity of the forkhead box transcription factor 3 (Foxp3)+ regulatory T (Treg) and conventional T (Tconv) cell populations active at sites of autoimmune pathology are not well characterized. To evaluate this, we combined global repertoire analyses and functional assessments of isolated T cell receptors (TCR) from TCRα retrogenic mice with autoimmune encephalomyelitis. Treg and Tconv cell TCR repertoires were distinct, and autoantigen-specific Treg and Tconv cells were enriched in diseased tissue. Autoantigen sensitivity and fine specificity of these cells intersected, implying that differences in responsiveness were not responsible for lineage specification. Notably, autoreactive Treg and Tconv cells could be fully distinguished by an acidic versus aliphatic variation at a single TCR CDR3 residue. Our results imply that ontogenically distinct Treg and Tconv cell repertoires with convergent specificities for autoantigen respond during autoimmunity and argue against more than limited plasticity between Treg and Tconv cells during autoimmune inflammation.
Previous genome-wide association studies for type 2 diabetes susceptibility genes have confirmed that a common variant, rs9939609, in the fat mass and obesity associated (FTO) gene region is associated with body mass index (BMI) in European children and adults. A significant association of the same risk allele has been described in Asian adult populations, but the results are conflicting. In addition, no replication studies have been conducted in children and adolescents of Asian ancestry.
A population-based survey was carried out among 3503 children and adolescents (6-18 years of age) in Beijing, China, including 1229 obese and 2274 non-obese subjects. We investigated the association of rs9939609 with BMI and the risk of obesity. In addition, we tested the association of rs9939609 with weight, height, waist circumference, waist-to-height ratio, fat mass percentage, birth weight, blood pressure and related metabolic traits.
We found significant associations of rs9939609 variant with weight, BMI, BMI standard deviation score (BMI-SDS), waist circumference, waist-to-height ratio, and fat mass percentage in children and adolescents (p for trend = 3.29 × 10-5, 1.39 × 10-6, 3.76 × 10-6, 2.26 × 10-5, 1.94 × 10-5, and 9.75 × 10-5, respectively). No significant associations were detected with height, birth weight, systolic and diastolic blood pressure and related metabolic traits such as total cholesterol, triglycerides, HDL-cholesterol, LDL-cholesterol and fasting plasma glucose (all p > 0.05). Each additional copy of the rs9939609 A allele was associated with a BMI increase of 0.79 [95% Confidence interval (CI) 0.47 to 1.10] kg/m2, equivalent to 0.25 (95%CI 0.14 to 0.35) BMI-SDS units. This rs9939609 variant is significantly associated with the risk of obesity under an additive model [Odds ratio (OR) = 1.29, 95% CI 1.11 to 1.50] after adjusting for age and gender. Moreover, an interaction between the FTO rs9939609 genotype and physical activity (p < 0.001) was detected on BMI levels, the effect of rs9939609-A allele on BMI being (0.95 ± 0.10), (0.77 ± 0.08) and (0.67 ± 0.05) kg/m2, for subjects who performed low, moderate and severe intensity physical activity.
The FTO rs9939609 variant is strongly associated with BMI and the risk of obesity in a population of children and adolescents in Beijing, China.