The next-generation antiandrogen MDV3100 prolongs overall survival of patients with metastatic castration-resistant prostate cancer (CRPC). However, patient responses are variable, and survival benefit remains relatively small. Developing effective modality to improve MDV3100 efficacy is urgently needed. Recent evidence suggests that constitutively active androgen receptor splice variants (AR-Vs) drive resistance to MDV3100. In our study, we show that methylselenol prodrug downregulates the expression and activity of both the full-length AR (AR-FL) and AR-Vs. The downregulation is independent of androgen and could be attributable to repressed transcription of the AR gene. Cotreatment with methylselenol prodrug and MDV3100 suppresses AR signaling more dramatically than either agent alone, and synergistically inhibits the growth of CRPC cells in vitro. The combinatorial efficacy is observed in not only AR-V-expressing cells but also cells expressing predominantly AR-FL, likely owing to the ability of the two drugs to block the AR signaling cascade at distinct steps. Ectopic expression of AR-FL or AR-V7 attenuates the combinatorial efficacy, indicating that downregulating AR-FL and AR-V7 is importantly involved in mediating the combinatorial efficacy. Significantly, methylselenol prodrug also downregulates AR-FL and AR-Vs in vivo and substantially improves the antitumor efficacy of MDV3100. These findings support a potential combination therapy for improving MDV3100 efficacy, and provide a rationale for evaluating the clinical application of combining methylselenol prodrug with MDV3100 for the treatment of CRPC.
methylseleninic acid; methylselenocysteine; MDV3100; androgen receptor; castration-resistant prostate cancer
Estrogen receptor α (ERα) plays a key role in the adaptation of increased uterine blood flow in pregnancy. Chronic hypoxia is a common stress to maternal cardiovascular homeostasis and causes increased risk of preeclampsia. Studies in pregnant sheep demonstrated that hypoxia during gestation downregulated ERα gene expression in uterine arteries. The present study tested the hypothesis that hypoxia causes epigenetic repression of the ERα gene in uterine arteries via heightened promoter methylation. Ovine ERα promoter of 2035 bp spanning from −2000 to +35 of the transcription start site was cloned. No estrogen or HIF response elements were found at the promoter. Two transcription factor binding sites USF-15 and Sp1-520 containing CpG dinucleotides were identified, which had significant effects on the promoter activity. The USF element binds transcription factors USF1 and USF2, and the Sp1 element binds Sp1, as well as ERα through Sp1. Deletion of the Sp1 site abrogated 17β-estradiol-induced increase in the promoter activity. In normoxic control sheep, CpG methylation at the Sp1, but not USF, site was significantly decreased in uterine arteries of pregnant, as compared with nonpregnant animals. In pregnant sheep exposed to long-term high altitude hypoxia, CpG methylation at both Sp1 and USF sites in uterine arteries was significantly increased. Methylation inhibited transcription factor binding and the promoter activity. The results provide evidence of hypoxia causing heightened promoter methylation and resultant ERα gene repression in uterine arteries, and suggest new insights of molecular mechanisms linking gestational hypoxia to aberrant uteroplacental circulation and increased risk of preeclampsia.
Hypoxia; estrogen receptor; methylation; epigenetic modulation; uterine artery
Although chronic hepatitis C virus (HCV) infection has been treated with the combination of interferon alpha (IFN-α) and ribavirin (RBV) for over a decade, the mechanism of antiviral synergy is not well understood. We aimed to determine the synergistic antiviral mechanisms of IFN-α and RBV combination treatment using HCV cell culture.
The antiviral efficacy of IFN-α, RBV alone and in combination was quantitatively measured using HCV infected and replicon cell culture. Direct antiviral activity of these two drugs at the level of HCV internal ribosome entry site (IRES) mediated translation in Huh-7 cell culture was investigated. The synergistic antiviral effect of IFN-α and RBV combination treatment was verified using both the CalcuSyn Software and MacSynergy Software.
RBV combination with IFN-α efficiently inhibits HCV replication cell culture. Our results demonstrate that IFN-α, interferon lambda (IFN-λ) and RBV each inhibit the expression of HCV IRES-GFP and that they have a minimal effect on the expression of GFP in which the translation is not IRES dependent. The combination treatments of RBV along with IFN-α or IFN-λ were highly synergistic with combination indexes <1. We show that IFN-α treatment induce levels of PKR and eIF2α phosphorylation that prevented ribosome loading of the HCV IRES-GFP mRNA. Silencing of PKR expression in Huh-7 cells prevented the inhibitory effect of IFN-α on HCV IRES-GFP expression. RBV also blocked polyribosome loading of HCV-IRES mRNA through the inhibition of cellular IMPDH activity, and induced PKR and eIF2α phosphorylation. Knockdown of PKR or IMPDH prevented RBV induced HCV IRES-GFP translation.
We demonstrated both IFN-α and RBV inhibit HCV IRES through prevention of polyribosome formation. The combination of IFN-α and RBV treatment synergistically inhibits HCV IRES translation via using two different mechanisms involving PKR activation and depletion of intracellular guanosine pool through inhibition of IMPDH.
Common variation at the loci harboring FTO, MC4R and TMEM18 is consistently reported as being statistically the most strongly associated with obesity. We investigated if these loci also harbor rarer missense variants that confer substantially higher risk of common childhood obesity in African American (AA) children. We sequenced the exons of FTO, MC4R and TMEM18 in an initial subset of our cohort i.e. 200 obese (BMI≥95th percentile) and 200 lean AA children (BMI≤5th percentile). Any missense exonic variants that were uncovered went on to be further genotyped in a further 768 obese and 768 lean (BMI≤50th percentile) children of the same ethnicity. A number of exonic variants were observed from our sequencing effort: seven in FTO, of which four were non-synonymous (A163T, G182A, M400V and A405V), thirteen in MC4R, of which six were non-synonymous (V103I, N123S, S136A, F202L, N240S and I251L) and four in TMEM18, of which two were non-synonymous (P2S and V113L). Follow-up genotyping of these missense variants revealed only one significant difference in allele frequency between cases and controls, namely with N240S in MC4R(Fisher's Exact P = 0.0001). In summary, moderately rare missense variants within the FTO, MC4R and TMEM18 genes observed in our study did not confer risk of common childhood obesity in African Americans except for a degree of evidence for one known loss-of-function variant in MC4R.
Obesity; Pediatrics; Genomics
Studies have indicated that the immune system plays a pivotal role in hepatitis. Substance P (SP) has been shown to modulate the immune response. In order to investigate the role of SP in liver injury and to determine whether it leads to pro-inflammatory signaling, we established a mouse model of hepatic injury induced by concanavalin A (ConA). We also exposed mouse Kupffer cells (KCs) to SP in vitro. Cytokine and SP levels in liver homogenates were detected using enzyme-linked immunosorbent assay (ELISA) and the protective effects of L-703,606 were evaluated through serological and histological assessments. Neurokinin-1 receptor (NK-1R) expression was evaluated by immunofluorescence and quantitative polymerase chain reaction (PCR). The levels of SP, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were significantly increased in the ConA-treated mice and the levels of ALT and AST were markedly reduced by L-703,606-pretreatment. Liver injury was significantly reduced by treatment with L-703,606. The mouse KCs expressed NK-1R and SP increased NK-1R mRNA expression. Furthermore, NK-1R blockade eliminated the effect of SP on NK-1R mRNA expression. The cytokine levels exhibited a substantial increase in the SP-pretreated KCs compared with the KCs that were cultured in control medium. The inter-leukin (IL)-6 and tumor necrosis factor (TNF)-α levels in the L-703,606-pretreated KCs were significantly lower compared with those in the SP-pretreated KCs. Our study suggests that neurogenic inflammation induced by SP plays an important role in hepatitis. Mouse KCs express NK-1R and SP increases NK-1R mRNA expression. SP enhances IL-6 and TNF-α secretion and an NK-1R antagonist inhibits this secretion.
concanavalin A; liver injury; substance P; neurokinin 1 receptor antagonist; neurogenic inflammation
Diabetic nephropathy (DN) is the most common cause of end-stage renal failure. Although angiotensin II receptor blockers (ARBs) can be used to attenuate proteinuria in DN patients, their efficacy remains limited. This clinical trial aimed to evaluate the efficacy of Tripterygium wilfordii Hook F (TwHF) extract in the treatment of type 2 diabetes mellitus (DM)-induced nephropathy.
A total of 65 DN patients with proteinuria levels ≥ 2.5 g/24 h and serum creatinine levels < 3 mg/dl were enrolled in this six-month, prospective, randomized, controlled study. The patients were randomized into treatment groups that received either 120 mg of TwHF extract per day for three months, followed by 60 mg per day for three more months, or 160 mg of valsartan daily for six months. The urinary protein and estimated glomerular filtration (eGFR) level were measured at one, three, and six months after the commencement of treatment. The primary measure of treatment efficacy was a reduction in the 24-h urine protein level between baseline and the end of the study, and the secondary measure of treatment efficacy was a reduction in the eGFR value.
At the end of the treatment period, the mean urine protein level in the TwHF group was dramatically decreased (4.99 ± 2.25 g/24 h vs 2.99 ± 1.81 g/24 h, p < 0.01), with decreases at one, three, and six months of 32.9%, 38.8%, and 34.3%, respectively. In contrast, the proteinuria in the valsartan group was not significantly attenuated, and the decreases in urine protein levels at treatment months one, three, and six were 1.05%, 10.1%, and -11.7%, respectively. The mean decrease in eGFR in the valsartan group was greater than that in the TwHF group (26.4% vs. 13.7%, respectively; p =0.067).
TwHF extract can reduce the urine protein level of DN patients and represents a novel, potentially effective, and safe drug for the treatment of DN patients with proteinuria.
Type 2 diabetes mellitus; Diabetic nephropathy; Proteinuria; Tripterygium wilfordii Hook F (TwHF); Angiotensin II receptor blocker (ARB)
The aim of this study was to compare the optic nerve head (ONH) and peripapillary retinal nerve fiber layer (RNFL) thickness in eyes with glaucoma and non-arteritic anterior ischemic neuropathy (NAION) by Fourier domain optical coherence tomography (FDOCT), and to evaluate the diagnostic capability of FDOCT in glaucoma and NAION. This study included 26 eyes with glaucoma (36.6%), 15 eyes with NAION (21.1%) and 30 eyes of normal subjects (42.3%). Those with the following conditions were excluded; a visual field defect greater than one hemifield, spherical equivalent (SE) more than ±6 D, or the onset of NAION within 6 months. FDOCT was used to analyze the characteristics of ONH and RNFL thickness. Among the three groups of subjects, glaucomatous eyes had the largest cup area and cup volume, and the smallest rim area, rim volume and disc volume (P<0.05). NAION eyes had the smallest cup area and cup volume (P<0.05), but their rim area, rim volume and disc volume were comparable to those of control eyes (P>0.05). The cup-to-disc (C/D) ratio was increased in glaucomatous eyes but reduced in NAION eyes compared with control eyes. Glaucomatous eyes had the greatest loss of RNFL thickness in the temporal upper (TU), superior temporal (ST) and temporal lower (TL) regions (P<0.05), whereas NAION eyes had the smallest RNFL thickness in the superior nasal (SN) and nasal upper (NU) regions (P<0.05). The areas under the receiver operator characteristic curve (AROCs) of the temporal, superior and inferior RNFL in glaucomatous eyes were greater compared with that of the disc area (P<0.05). In addition, the AROCs of the temporal, superior and inferior RNFL were higher compared with that of nasal RNFL (P<0.05). The AROCs of all parameters for NAION were not significantly different, with the exception of superior, nasal superior and inferior temporal RNFL (P<0.05). In conclusion, FDOCT is able to detect quantitative differences in the optic disc morphology and RNFL thickness between glaucomatous and NAION eyes. These differences may provide new insights into the clinical characteristics and diagnosis of the two diseases.
optic disc; retinal nerve fiber layer; glaucoma; ischemic optic neuropathy; optic coherence tomography
Neuroblastoma (NBL) is an often-fatal pediatric cancer more frequent in European-American than African-American children. African-American children, however, are at higher risk for the more severe form of NBL, and have worse overall survival than European-American children. Genome wide association studies (GWAS) have identified several SNPs associated to NBL in children of European descent. Knowledge of their association to NBL in African-American children is still lacking.
We genotyped and imputed SNPs located in three gene regions reported to be associated to NBL in children of European descent, and tested them for association in 390 African-American NBL patients compared to 2500 healthy, ethnically matched controls.
SNPs in the BARD1 gene region show a similar pattern of association to NBL in African-American and European-American children. The more restricted extent of linkage disequilibrium in the African-American population suggests a smaller candidate region for the putative causal variants than previously reported. Limited association was observed at the other two gene regions tested, including LMO1 in 11p15 and FLJ22536 in 6p22.
Common BARD1 SNPs affect risk of NBL in African-Americans. The role of other SNPs associated to NBL in children of European descent could not be confirmed, possibly due to different patterns of linkage disequilibrium or limited statistical power to detect association to variants with small effect on disease risk. Extension of GWAS to populations of African descent is important to confirm their results and validity beyond the European populations, and can help to refine the location of the putative causal variants.
Neuroblastoma; BARD1; African-Americans; SNPs; genetic association
Protein kinase Cε (PKCε) plays a pivotal role in cardioprotection during cardiac ischemia and reperfusion injury. Recent studies demonstrated that prenatal cocaine exposure caused a decrease in PKCε expression and increased heart susceptibility to ischemic injury in adult offspring, suggesting an in utero programming of PKCε gene expression pattern in the heart. The present investigation aimed to elucidate whether an epigenetic mechanism, DNA methylation, accounts for cocaine-mediated repression of the PKCε gene in the heart. Pregnant rats were administered either saline or cocaine intraperitoneally (15 mg/kg) twice daily from days 15 to 20 of gestational age, and term fetal hearts were studied. Cocaine treatment significantly decreased PKCε mRNA and protein levels in the heart. CpG dinucleotides found in cAMP response element-binding protein (CREB), CREB/c-Jun1, and CREB/c-Jun2 binding sites at the proximal promoter region of the PKCε gene were densely methylated and were not affected by cocaine. In contrast, methylation of CpGs in the activator protein 1 (AP-1) binding sites was low but was significantly increased by cocaine. Reporter gene assays showed that the AP-1 binding site played a strong stimulatory role of PKCε gene transcription. Methylation of the AP-1 binding sites significantly decreased AP-1 binding to the PKCε promoter. Supershift analyses implicated c-Jun homodimers binding to the AP-1 binding sites. Cocaine did not affect nuclear c-Jun levels or the binding of c-Jun to the unmethylated AP-1 binding sites. The results indicate a role for DNA methylation in cocaine-mediated PKCε gene repression in the developing heart and suggest an epigenetic mechanism affecting this gene linked with vulnerability of ischemic injury in the heart of adult offspring.
A thorough understanding of the mechanisms underlying barley salt tolerance and exploitation of elite genetic resource are essential for utilizing wild barley germplasm in developing barley varieties with salt tolerance. In order to reveal the physiological and molecular difference in salt tolerance between Tibetan wild barley (Hordeum spontaneum) and cultivated barley (Hordeum vulgare), profiles of 82 key metabolites were studies in wild and cultivated barley in response to salinity. According to shoot dry biomass under salt stress, XZ16 is a fast growing and salt tolerant wild barley. The results of metabolite profiling analysis suggested osmotic adjustment was a basic mechanism, and polyols played important roles in developing salt tolerance only in roots, and high level of sugars and energy in roots and active photosynthesis in leaves were important for barley to develop salt tolerance. The metabolites involved in tolerance enhancement differed between roots and shoots, and also between genotypes. Tibetan wild barley, XZ16 had higher chlorophyll content and higher contents of compatible solutes than CM72, while the cultivated barley, CM72 probably enhanced its salt tolerance mainly through increasing glycolysis and energy consumption, when the plants were exposed to high salinity. The current research extends our understanding of the mechanisms involved in barley salt tolerance and provides possible utilization of Tibetan wild barley in developing barley cultivars with salt tolerance.
Hepatitis viral B x protein (HBx), a hepatocarcinogen, is frequently mutated. Hypoxia influences the growth of HCC and also the sensitivity of tumor cells to treatments. We aimed to test the role of HBx and acute hypoxia in the efficacy of chemotherapy. In this study, we established 4 Chang liver cell lines with the full-length HBx (HBx), the first 50 amino acids of N-terminal HBx (HBx/50), the last 104 amino acids of C-terminal HBx (HBx/51) and empty vector (CL), respectively. MTT and TNUEL assays were used to assess cell viability and apoptosis respectively. Western blot was used to determine the expression of relevant proteins. Results showed that among 4 cell lines, doxorubicin was most effective in decreasing the viability and enhancing apoptosis in HBx/51 cells, while HBx/50 cells were most resistant to the treatment. Cells in hypoxia were more susceptible to doxorubicin than cells in normoxia. Hypoxia facilitated the Bid cleavage especially in HBx/51 cells via phosphorylating p38 MAPK. p38 MAPK inhibitor significantly reduced the tBid level and increased cell viability. In conclusion, N-terminal HBx and C-terminal HBx function differentially in their ability to regulate cell growth, with the former being promotive but the latter being inhibitory. The acute hypoxia may overcome the HBx-induced resistance and facilitate the chemotherapy.
The androgen receptor (AR) is critical in the normal development and function of the prostate, as well as in prostate carcinogenesis. Androgen deprivation therapy is the mainstay in the treatment of advanced prostate cancer, however, after an initial response, the disease inevitably progresses to castration-resistant prostate cancer (CRPC). Recent evidence suggests that continued AR activation, sometimes in a ligand-independent manner, is commonly associated with the development of CRPC. Thus, novel agents targeting the AR are urgently needed as a strategic step in developing new therapies for this disease state. In this study, we investigated the effect of berberine on AR signaling in prostate cancer. We report that berberine decreased the transcriptional activity of AR. Berberine did not affect AR mRNA expression, but induced AR protein degradation. Several ligand-binding domain truncated AR splice variants have been identified and these variants are believed to promote the development of CRPC in patients. Interestingly, we found that these variants were more susceptible to berberine-induced degradation than the full-length AR. Furthermore, the growth of LNCaP xenografts in nude mice was inhibited by berberine and AR expression was reduced in the tumors, whereas the morphology and AR expression in normal prostates were not affected. This report is the first to show that berberine suppresses AR signaling and suggests that berberine or its derivatives is a promising agent for the prevention and/or treatment of prostate cancer.
berberine; androgen receptor; prostate cancer; xenograft
Finasteride is known to inhibit Type 2 5α-reductase and thus block the conversion of testosterone to dihydrotestosterone (DHT). The structural similarity of finasteride to DHT raises the possibility that finasteride may also interfere with the function of the androgen receptor (AR). Experiments were carried out to evaluate the antiandrogenic effect of finasteride in LNCaP, C4-2 and VCaP human prostate cancer cells. Finasteride decreased DHT binding to AR, and DHT-stimulated AR activity and cell growth in LNCaP and C4-2 cells, but not in VCaP cells. LNCaP and C4-2 (derived from castration-resistant LNCaP) cells express the T877A mutant AR, while VCaP cells express the wild-type AR. When PC-3 cells, which are AR-null, were transfected with either the wild-type or the T877A mutant AR, only the mutant AR-expressing cells were sensitive to finasteride inhibition of DHT binding. Peroxiredoxin-1 (Prx1) is a novel endogenous facilitator of AR binding to DHT. In Prx1-rich LNCaP cells, the combination of Prx1 knockdown and finasteride was found to produce a greater inhibitory effect on AR activity and cell growth than either treatment alone. The observation suggests that cells with a low expression of Prx1 are likely to be more responsive to the antiandrogenic effect of finasteride. Additional studies showed that the efficacy of finasteride was comparable to that of bicalutamide (a widely used non-steroidal antiandrogen). The implication of the above findings is discussed in the context of developing strategies to improve the outcome of androgen deprivation therapy.
finasteride; androgen receptor; prostate cancer; antagonistic effect; peroxiredoxin-1; DHT; mutant AR
Neuroblastoma is a childhood cancer of the sympathetic nervous system that accounts for approximately 10% of all paediatric oncology deaths1,2. To identify genetic risk factors for neuroblastoma, we performed a genome-wide association study (GWAS) on 2,251 patients and 6,097 control subjects of European ancestry from four case series. Here we report a significant association within LIM domain only 1 (LMO1) at 11p15.4 (rs110419, combined P = 5.2 × 10−16, odds ratio of risk allele = 1.34 (95% confidence interval 1.25–1.44)). The signal was enriched in the subset of patients with the most aggressive form of the disease. LMO1 encodes a cysteine-rich transcriptional regulator, and its paralogues (LMO2, LMO3 and LMO4) have each been previously implicated in cancer. In parallel, we analysed genome-wide DNA copy number alterations in 701 primary tumours. We found that the LMO1 locus was aberrant in 12.4% through a duplication event, and that this event was associated with more advanced disease (P < 0.0001) and survival (P = 0.041). The germline single nucleotide polymorphism (SNP) risk alleles and somatic copy number gains were associated with increased LMO1 expression in neuroblastoma cell lines and primary tumours, consistent with a gain-of-function role in tumorigenesis. Short hairpin RNA (shRNA)-mediated depletion of LMO1 inhibited growth of neuroblastoma cells with high LMO1 expression, whereas forced expression of LMO1 in neuroblastoma cells with low LMO1 expression enhanced proliferation. These data show that common polymorphisms at the LMO1 locus are strongly associated with susceptibility to developing neuroblastoma, but also may influence the likelihood of further somatic alterations at this locus, leading to malignant progression.
Glucose transporter 4 (GLUT4) is a principal glucose transporter in response to insulin, and impaired translocation or decreased expression of GLUT4 is believed to be one of the major pathological features of type 2 diabetes mellitus (T2DM). Therefore, induction of GLUT4 translocation or/and expression is a promising strategy for anti-T2DM drug discovery. Here we report that the natural product (+)-Rutamarin (Rut) functions as an efficient dual inducer on both insulin-induced GLUT4 translocation and expression. Rut-treated 3T3-L1 adipocytes exhibit efficiently enhanced insulin-induced glucose uptake, while diet-induced obese (DIO) mice based assays further confirm the Rut-induced improvement of glucose homeostasis and insulin sensitivity in vivo. Subsequent investigation of Rut acting targets indicates that as a specific protein tyrosine phosphatase 1B (PTP1B) inhibitor Rut induces basal GLUT4 translocation to some extent and largely enhances insulin-induced GLUT4 translocation through PI3 kinase-AKT/PKB pathway, while as an agonist of retinoid X receptor α (RXRα), Rut potently increases GLUT4 expression. Furthermore, by using molecular modeling and crystallographic approaches, the possible binding modes of Rut to these two targets have been also determined at atomic levels. All our results have thus highlighted the potential of Rut as both a valuable lead compound for anti-T2DM drug discovery and a promising chemical probe for GLUT4 associated pathways exploration.
A major challenge in breast cancer therapy is the lack of an effective therapeutic option for a particularly aggressive subtype of breast cancer, triple-negative breast cancer. Here we provide the first preclinical evidence that a second-generation selenium compound, methylseleninic acid, significantly enhances the anticancer efficacy of paclitaxel in triple-negative breast cancer. Through combination-index value calculation, we demonstrated that methylseleninic acid synergistically enhanced the growth inhibitory effect of paclitaxel in triple-negative breast cancer cells. The synergism was attributable to more pronounced induction of caspase-mediated apoptosis, arrest of cell cycle progression at the G2/M checkpoint, and inhibition of cell proliferation. Treatment of SCID mice bearing MDA-MB-231 triple-negative breast cancer xenografts for four weeks with methylseleninic acid (4.5 mg/kg/day, orally) and paclitaxel (10 mg/kg/week, through intraperitoneal injection) resulted in a more pronounced inhibition of tumor growth compared with either agent alone. The attenuated tumor growth correlated with a decrease in tumor cell proliferation and an induction of apoptosis. The in vivo study also indicated the safety of using methylseleninic acid in the combination regime. Our findings thus provide strong justification for the further development of methylseleninic acid and paclitaxel combination therapy for the treatment of triple-negative breast cancer.
Saccharophagus degradans is an aerobic marine bacterium that can degrade cellulose by the induced expression of an unusual cellulolytic system composed of multiple endoglucanases and glucosidases. To understand the regulation of the cellulolytic system, transcript levels for the genes predicted to contribute to the cellulolytic system were monitored by quantitative real-time PCR (qRT-PCR) during the transition to growth on cellulose. Four glucanases of the cellulolytic system exhibited basal expression during growth on glucose. All but one of the predicted cellulolytic system genes were induced strongly during growth on Avicel, with three patterns of expression observed. One group showed increased expression (up to 6-fold) within 4 h of the nutritional shift, with the relative expression remaining constant over the next 22 h. A second group of genes was strongly induced between 4 and 10 h after nutritional transfer, with relative expression declining thereafter. The third group of genes was slowly induced and was expressed maximally after 24 h. Cellodextrins and cellobiose, products of the predicted basally expressed endoglucanases, stimulated expression of representative cellulase genes. A model is proposed by which the activity of basally expressed endoglucanases releases cellodextrins from Avicel that are then perceived and transduced to initiate transcription of each of the regulated cellulolytic system genes forming an expression pattern.
The inflammatory bowel diseases (IBD) Crohn’s disease and ulcerative colitis are common causes of morbidity in children and young adults in the western world. Here we report the results of a genome-wide association study in early-onset IBD involving 3,426 affected individuals and 11,963 genetically matched controls recruited through international collaborations in Europe and North America, thereby extending the results from a previous study of 1,011 individuals with early-onset IBD1. We have identified five new regions associated with early-onset IBD susceptibility, including 16p11 near the cytokine gene IL27 (rs8049439, P = 2.41 × 10−9), 22q12 (rs2412973, P = 1.55 × 10−9), 10q22 (rs1250550, P = 5.63 × 10−9), 2q37 (rs4676410, P = 3.64 × 10−8) and 19q13.11 (rs10500264, P = 4.26 × 10−10). Our scan also detected associations at 23 of 32 loci previously implicated in adult-onset Crohn’s disease and at 8 of 17 loci implicated in adult-onset ulcerative colitis, highlighting the close pathogenetic relationship between early- and adult-onset IBD.
Foetal nicotine exposure results in decreased protein kinase C epsilon (PKCε) expression and increased cardiac vulnerability to ischaemia and reperfusion injury in adult rat offspring. The present study tested the hypothesis that maternal nicotine administration causes increased promoter methylation of the PKCε gene resulting in PKCε repression in the heart.
Methods and results
Nicotine treatment of pregnant rats starting at day 4 of gestation increased the methylation of the Egr-1 binding site at the PKCε gene promoter and decreased PKCε protein and mRNA abundance in near-term foetal hearts. Methylation of the Egr-1 binding site reduced Egr-1 binding to the PKCε promoter in the heart. Site-specific deletion of the Egr-1 binding site significantly decreased PKCε promoter activity. The effects of nicotine were sustained in the heart of adult offspring. Ex vivo studies found no direct effect of nicotine on PKCε gene expression. However, maternal nicotine administration increased norepinephrine content in the foetal heart. Treatment of isolated foetal hearts with norepinephrine resulted in the same effects of increased methylation of the Egr-1 binding site and PKCε gene repression in the heart. 5-Aza-2′-deoxycytidine inhibited the norepinephrine-induced increase in methylation of the Egr-1 binding site and restored Egr-1 binding and PKCε gene expression to the control levels.
This study demonstrates that prolonged nicotine exposure increases the sympathetic neurotransmitter release in the foetal heart and causes programming of PKCε gene repression through promoter methylation, linking maternal smoking to pathophysiological consequences in the offspring heart.
Nicotine; Heart; Norepinephrine; Protein kinase C; DNA methylation
Nuclear receptors retinoic X receptor α (RXRα) and peroxisome proliferator activated receptor γ (PPARγ) function potently in metabolic diseases, and are both important targets for anti-diabetic drugs. Coactivation of RXRα and PPARγ is believed to synergize their effects on glucose and lipid metabolism. Here we identify the natural product magnolol as a dual agonist targeting both RXRα and PPARγ. Magnolol was previously reported to enhance adipocyte differentiation and glucose uptake, ameliorate blood glucose level and prevent development of diabetic nephropathy. Although magnolol can bind and activate both of these two nuclear receptors, the transactivation assays indicate that magnolol exhibits biased agonism on the transcription of PPAR-response element (PPRE) mediated by RXRα:PPARγ heterodimer, instead of RXR-response element (RXRE) mediated by RXRα:RXRα homodimer. To further elucidate the molecular basis for magnolol agonism, we determine both the co-crystal structures of RXRα and PPARγ ligand-binding domains (LBDs) with magnolol. Structural analyses reveal that magnolol adopts its two 5-allyl-2-hydroxyphenyl moieties occupying the acidic and hydrophobic cavities of RXRα L-shaped ligand-binding pocket, respectively. While, two magnolol molecules cooperatively accommodate into PPARγ Y-shaped ligand-binding pocket. Based on these two complex structures, the key interactions for magnolol activating RXRα and PPARγ are determined. As the first report on the dual agonist targeting RXRα and PPARγ with receptor-ligand complex structures, our results are thus expected to help inspect the potential pharmacological mechanism for magnolol functions, and supply useful hits for nuclear receptor multi-target ligand design.
Rice Xa3/Xa26 disease-resistance gene encodes a leucine-rich repeat (LRR) receptor kinase-type protein against Xanthomonas oryzae pv. oryzae (Xoo) and belongs to a multigene family. However, the functions of most genes in this family are unknown.
Here we report that two orthologs of this family, the NRKe from rice variety Nipponbare and 9RKe from variety 93-11 at the RKe locus, have similar functions although they encode different proteins. This pair of orthologs could not mediate resistance to Xoo, but they were transcriptionally induced by raised temperature. Transcriptional activation of NRKe or 9RKe resulted in the formation of temperature-sensitive lesion mimics, which were spots of dead cells associated with accumulation of superoxides, in different organs of the transgenic plants. These plants were more sensitive to high temperature shock than wild-type controls. Transgenic plants carrying a chimeric protein consisting of the LRR domain of NRKe and the kinase domain of Xa3/Xa26 developed the same lesion mimics as the NRKe-transgenic plants, whereas transgenic plants carrying another chimeric protein consisting of the LRR domain of Xa3/Xa26 and the kinase domain of NRKe were free of lesion mimic. All the transgenic plants carrying a chimeric protein were susceptible to Xoo.
These results suggest that the RKe locus is involved in rice response to raised temperature. The LRR domain of RKe protein appears to be important to sense increased temperature. The RKe-involved temperature-related pathway and Xa3/Xa26-mediated disease-resistance pathway may partially overlap.
Diabetes impacts approximately 200 million people worldwide, of whom approximately 10% are affected by type 1 diabetes (T1D). The application of genome-wide association studies (GWAS) has robustly revealed dozens of genetic contributors to the pathogenesis of T1D, with the most recent meta-analysis identifying in excess of 40 loci. To identify additional genetic loci for T1D susceptibility, we examined associations in the largest meta-analysis to date between the disease and ∼2.54 million SNPs in a combined cohort of 9,934 cases and 16,956 controls. Targeted follow-up of 53 SNPs in 1,120 affected trios uncovered three new loci associated with T1D that reached genome-wide significance. The most significantly associated SNP (rs539514, P = 5.66×10−11) resides in an intronic region of the LMO7 (LIM domain only 7) gene on 13q22. The second most significantly associated SNP (rs478222, P = 3.50×10−9) resides in an intronic region of the EFR3B (protein EFR3 homolog B) gene on 2p23; however, the region of linkage disequilibrium is approximately 800 kb and harbors additional multiple genes, including NCOA1, C2orf79, CENPO, ADCY3, DNAJC27, POMC, and DNMT3A. The third most significantly associated SNP (rs924043, P = 8.06×10−9) lies in an intergenic region on 6q27, where the region of association is approximately 900 kb and harbors multiple genes including WDR27, C6orf120, PHF10, TCTE3, C6orf208, LOC154449, DLL1, FAM120B, PSMB1, TBP, and PCD2. These latest associated regions add to the growing repertoire of gene networks predisposing to T1D.
Despite the fact that there is clearly a large genetic component to type 1 diabetes (T1D), uncovering the genes contributing to this disease has proven challenging. However, in the past three years there has been relatively major progress in this regard, with advances in genetic screening technologies allowing investigators to scan the genome for variants conferring risk for disease without prior hypotheses. Such genome-wide association studies have revealed multiple regions of the genome to be robustly and consistently associated with T1D. More recent findings have been a consequence of combining of multiple datasets from independent investigators in meta-analyses, which have more power to pick up additional variants contributing to the trait. In the current study, we describe the largest meta-analysis of T1D genome-wide genotyped datasets to date, which combines six large studies. As a consequence, we have uncovered three new signals residing at the chromosomal locations 13q22, 2p23, and 6q27, which went on to be replicated in independent sample sets. These latest associated regions add to the growing repertoire of gene networks predisposing to T1D.
The evaluation of both the genetic variation and the identification of salinity tolerant accessions of Tibetan annual wild barley (hereafter referred to as Tibetan barley) (Hordeum vulgare L. ssp. Spontaneum and H. vulgare L. ssp. agriocrithum) are essential for discovering and exploiting novel alleles involved in salinity tolerance. In this study, we examined tissue dry biomass and the Na+ and K+ contents of 188 Tibetan barley accessions in response to salt stress. We investigated the genetic variation of transcription factors HvCBF1, HvCBF3 and HvCBF4 within these accessions, conducting association analysis between these three genes and the respective genotypic salt tolerance. Salt stress significantly reduced shoot and root dry weight by 27.6% to 73.1% in the Tibetan barley lines. HvCBF1, HvCBF3 and HvCBF4 showed diverse sequence variation in amplicon as evident by the identification of single nucleotide polymorphisms (SNPs) and 3, 8 and 13 haplotypes, respectively. Furthermore, the decay of Linkage disequilibrium (LD) of chromosome 5 was 8.9 cM (r2<0.1). Marker bpb-4891 and haplotype 13 (Ps 610) of the HvCBF4 gene were significantly (P<0.05) and highly significantly (P<0.001) associated with salt tolerance. However, HvCBF1 and HvCBF3 genes were not associated with salinity tolerance. The accessions from haplotype 13 of the HvCBF4 gene showed high salinity tolerance, maintaining significantly lower Na+/K+ ratios and higher dry weight. It is thus proposed that these Tibetan barley accessions could be of value for enhancing salinity tolerance in cultivated barley.
As the mainstay treatment for advanced prostate cancer, androgen-deprivation therapy (ADT) targets the action of androgen receptor (AR) by reducing androgen level and/or by using anti-androgen to compete with androgens for binding to AR. Albeit effective in extending survival, ADT is associated with dose-limiting toxicity and the development of castration-resistant prostate cancer (CRPC) after prolonged use. Since CRPC is lethal and incurable, developing effective strategies to enhance the efficacy of ADT and circumvent resistance becomes an urgent task. Continuous AR signaling constitutes one major mechanism underlying the development of CRPC. The present study showed that methylseleninic acid (MSA), an agent that effectively reduces AR abundance, could enhance the cancer-killing efficacy of the anti-androgen bicalutamide in both androgen-dependent and castration-resistant prostate cancer cells. We found that combination of MSA and bicalutamide produced a robust downregulation of prostate-specific antigen and a recently identified AR target, telomerase and its catalytic subunit, telomere reverse transcriptase (hTERT). The downregulation of hTERT occurs mainly at the transcriptional level, and reduced AR occupancy of the promoter contributes to the downregulation. Furthermore, apoptosis induction by the two agents is significantly mitigated by restoration of hTERT. Our findings thus indicate that MSA in combination with anti-androgen could represent a viable approach to improve the therapeutic outcome of ADT. Given the critical role of hTERT/telomerase downregulation in mediating the combination effect and the fact that hTERT/telomerase could be measured in blood and urine, hTERT/telomerase could serve as an ideal tumor-specific biomarker to monitor the efficacy of the combination therapy non-invasively.
anti-androgen; methylseleninic acid; androgen receptor; telomerase; prostate cancer
Inflammatory bowel disease, including Crohn's disease (CD) and ulcerative colitis (UC), and type 1 diabetes (T1D) are autoimmune diseases that may share common susceptibility pathways. We examined known susceptibility loci for these diseases in a cohort of 1689 CD cases, 777 UC cases, 989 T1D cases and 6197 shared control subjects of European ancestry, who were genotyped by the Illumina HumanHap550 SNP arrays. We identified multiple previously unreported or unconfirmed disease associations, including known CD loci (ICOSLG and TNFSF15) and T1D loci (TNFAIP3) that confer UC risk, known UC loci (HERC2 and IL26) that confer T1D risk and known UC loci (IL10 and CCNY) that confer CD risk. Additionally, we show that T1D risk alleles residing at the PTPN22, IL27, IL18RAP and IL10 loci protect against CD. Furthermore, the strongest risk alleles for T1D within the major histocompatibility complex (MHC) confer strong protection against CD and UC; however, given the multi-allelic nature of the MHC haplotypes, sequencing of the MHC locus will be required to interpret this observation. These results extend our current knowledge on genetic variants that predispose to autoimmunity, and suggest that many loci involved in autoimmunity may be under a balancing selection due to antagonistic pleiotropic effect. Our analysis implies that variants with opposite effects on different diseases may facilitate the maintenance of common susceptibility alleles in human populations, making autoimmune diseases especially amenable to genetic dissection by genome-wide association studies.