In the preschool period, allergic rhinitis (AR) is infrequent and thus under-diagnosed. However, recent works have highlighted the occurrence of AR in toddlers although the causes of AR in this young population remain unknown. The objective of this study was to identify determinants of AR in young children with asthma.We carried out a case-control study of 227 children with active asthma and enrolled in the Trousseau Asthma Program. AR and other allergic diseases  were diagnosed by medical doctors using standardized questionnaires. Parental history of AR and asthma, biological markers of atopy  and environmental parameters were also collected.Forty one of the children (18.1%) had AR. By univariate logistic regression analysis, AR was mainly associated with peanut sensitization ; food allergy ; mold exposure (OR = 3.81 p<0.01) and parental history of AR . Due to the strong link between food allergy and peanut sensitization three models of multivariate logistic regression were performed and confirmed that AR is associated with peanut sensitization but also food allergy and mold exposure. A random forest analysis was also performed to explain AR. The results reinforced the logistic analysis that peanut sensitization and mold exposure were the principal determinants of AR.These results stress the importance of investigating AR in young children with asthma to potentially diagnose a particularly severe allergic asthmatic phenotype. Moreover, these data evoke the hypothesis that peanut could be an aeroallergen. Allergic rhinitis (AR) is an atopic manifestation frequently associated with asthma. The prevalence of AR in children has been rising in many countries and is associated with a western lifestyle The « Pollution and Asthma Risk: an Infant Study » (PARIS) birth cohort, gave an estimated general prevalence of AR of around 9.1% in 18-month-old infants Our study set out to identify determinants associated with AR in a case-control study performed in a cohort of young children with asthma.Data were collected by standardized questionnaires and the medical examination was conducted by a physician after the parents gave their written informed consent. The Ile de France V ethics committee approved the protocol and this consent procedure.This case-control study gathered data from January to November 2011 and enrolled children who were part of the TAP (Trousseau Asthma Program). All the children had been referred to the center for recurrent wheezing by a primary care physician and joined the study consecutively.Inclusion criteria for the study were: age younger than 30 months; persistent asthma  General data were collected on the children's gender and age.AR was diagnosed by a medical doctor supported by the SFAR criteria as defined by a score of 7 or more Personal history of atopy (eczema) was mostly assessed by questions from the International Study of Asthma and Allergies in Childhood (ISAAC) Moderate to severe asthma was determined according to the National Asthma Education and Prevention Program Finally, information about parental history  of AR and asthma were also gathered.2 for each person living at home). Potential sources of biologic allergens were also evaluated: mold exposure (visible mold and moldy smell) and presence of visible cockroaches at home.Environmental factors were assessed by the questions applied in the PARIS birth cohort. We collected data about exclusive maternal breast-feeding beyond 3 months, exposure to passive smoking (defined by smokers at home smoking more than five cigarettes a day) and overcrowding , increased total IgE as a concentration ≥45 kU/L Biological markers of atopy were measured in peripheral blood. These included the multiple allergenic screening test Phadiatop infant and blood levels of specific IgE toward inhaled and food allergens  for children testing positive. Children with two or more sensitizations to food and/or aeroallergens were considered to have polysensitization. Aeroallergen sensitization was defined as at least one sensitization to an inhaled allergen. Other inflammatory biological markers, such as total eosinophilia count and serum level of total IgE, were measured too. Thresholds were used to define increased levels: increased blood eosinophilia was defined as a concentration ≥470 eosinophils/mmThe chi-square test and Fisher's exact test were used to compare the distribution of each variable  between the control and the AR group. A logistic regression analysis http://www.r-project.org). The R package ‘glm’, ‘Rpart’ and ‘randomForest’ were used to perform the analysis.A random forest analysis Forty-one of the 227 children seen in the TAP between January and November 2011 (18.1%) had AR. Among the remaining 186 asthmatic children without AR, 88 were randomized and enrolled in the control group. The two groups were similar regarding age , allergic polysensitization (p = 0.030), especially due to peanut sensitization (p = 0.002) and greater exposure to mold (p = 0.030). Manifestations of food allergy were cutaneous  for seven of them and digestive (nausea or vomiting) for three. One child had both types of symptoms.In univariate logistic regression analysis, AR was found to be mainly associated with food allergy ; peanut  and then with one environmental factor: mold exposure . Other risk factors were found to be: biological biomarkers identified by Phadiatop infant , more precisely polysensitization ; and egg sensitization ; and finally a parental history of AR .a = 3.96; p = 0.081), peanut sensitization , mold exposure , familial history of AR  and moderate to severe asthma  (model 1). A correlation study highlighted the association between food allergy and peanut sensitization . Such a correlation should be avoided as the two variables share common information. We thus constructed a model without peanut sensitization (Model 2) and a model without food allergy (Model 3)  . The OR The importance measure (permutation measure) obtained from the random forest analysis  can be iThe main result of our study is that independent variables for AR in young asthmatic children are mainly peanut sensitization  and a moldy environment. Taking together all the results, in a less constant manner, an association was found between AR and moderate to severe asthma but also a parental history of AR and polysensitization.et al.In school age children, co-morbidities of allergic diseases such as AR and asthma occur more frequently in sensitized children  et al. highlighted the fact that sensitization to a food allergen is a risk factor for further sensitization to an aeroallergen even without clinical food allergy In our study, peanut sensitization appeared to be an important determinant of AR in preschool children. Similarly, Kulig Moreover, we showed a significant link between food allergy and AR within the same toddler period. The relatively low importance of food allergy in random forest analysis was explained by the correlation with peanut sensitization de novo development of AR  and showed the increased risk to be dose-dependent Our findings identified indoor dampness and the resulting molds as important risk factors for AR in young children with asthma. This risk has also been pointed out by the ISAAC study in the 6 to 7 year-old age group Our study shows a link between the development of AR in early childhood and a parental history of AR. This result is in line with previous findings. The follow-up of the PARIS birth cohort estimated that a double parental history is associated with a twofold increased risk of the infant developing AR. In a study of children in Singapore, the risk of AR was 4.5 times higher (CI 95%: 3.3–6.1) when both parents had AR A link between AR and moderate to severe asthma as independent variables was found by random forest analysis with a borderline association in the three multivariate logistic regression models. It has been well reported in the literature that AR worsens the asthma prognosis et al.Simpson et al.On a clinical level, Schröder et al.The main strength of this study lies in the use of precisely defined clinical parameters and biological markers. AR was assessed by medical doctors, supported by the SFAR criteria, which were more stringent than those used in previous studies Our study reveals that in young children with asthma, AR could be a signal symptom of a severe allergic phenotype with a particular association with peanut sensitization. This data stresses the importance of thoroughly investigating AR so as not to miss potentially diagnosing a particular severe asthmatic phenotype. Finally our results raise the hypothesis that peanut could be considered as an inhaled allergen.


The objective of this study was to investigate the relationship between prenatal and postnatal risk factors and the prevalence of allergic rhinitis (AR) in Chinese children of specific ages.This study was a cross-sectional survey. Students from 8 metropolitan cities in China were studied in November and December, 2005. There were 20,803 elementary-school Chinese children  enrolled. Questions from the standard questionnaire of the International Study of Asthma and Allergies in Children were completed to enable us to examine the pattern of current AR. The potential confounders and pre-and postnatal risk factors were analyzed using logistic regression.The overall prevalence of AR was found in this study to be 9.8%. After adjusting for several likely confounders, there was a higher likelihood of AR in school-aged children who were not exclusively breastfed in the first 4 months of their lives , children who were born post-term , children delivered by cesarean section , or children born to mothers who experienced depressive symptoms during the pre- and postnatal periods .AR in school-aged children was found to be associated with pre- and postnatal events. These findings indicate that strategies to reduce exposure to risk factors during pre- and postnatal periods for childhood allergies might be warranted. Allergic rhinitis (AR), a chronic inflammatory disease of the upper airway, is not only one of the most common chronic diseases in children, but also a global health problem . AR is cAlthough the etiology of AR is uncertain, there is evidence to suggest that an atopic phenotype might be programmed in-utero . InteracA national survey on children’s sleep patterns was conducted in eight Chinese cities in 2005 . BecauseThe study based on childhood sleep patterns was conducted between November and December 2005 in eight cities in China through a cluster-stratified selection. The surveys were performed in areas of northern, southern, and central China, comprising Hohhot (HO), Urumqi (UR), Harbin (HA), Shanghai (SH), Guangzhou (GZ), Xi’an (XA), Wuhan (WH) and Chengdu (CD). These eight cities were chosen because they have high levels of health resources and public awareness about childhood allergic diseases, and doctors generally follow the national standard for the clinical diagnosis of AR. These cities were divided into two groups for this study according to their gross domestic product (GDP) and health-system coverage . Those wThe study population comprised 5- to 13-year-old children attending the first to sixth grades of primary school. 3–10 districts were randomly selected for each city and within each district, 1–2 elementary schools were chosen. Thirty districts and 42 schools in urban areas, 9 districts and 13 schools in suburban areas constituted the final sample.Rhinitis and asthma are linked by epidemiological, pathological and physiological characteristics and by a common therapeutic approach. According to the concept of “one airway, one disease” for asthma and rhinitis , 716 astStudy design, enrollment, criteria for inclusion and procedures for data collection were described in detail in previous articles . In brieAtopic disease questionnaires. Key questions were taken from the Chinese version of the ISAAC questionnaire, which exhibited good reliability and validity to examine the prevalence of current AR to compare with results in the literature [terature . The queSociodemographic questionnaires. The sociodemographic questionnaire surveyed for factors that might have had a significant impact on AR. These comprised the following variables: gender, age, gestational age (<37 weeks/ 37–41 weeks/≥42 weeks), method of delivery , exclusive breastfeeding in the first 4 months (yes/no), parental educational level (middle school and below/high school/college and above), family income (yuan/month/person), resident area per captia (m2), family numbers (<3 persons/3 persons/≥ persons), mother having depressive symptoms during pre- and postnatal period (yes/no).Statistical analysis was performed including the use of the mean and standard deviation (SD) for continuous variables, and percentages for categorical variables. Firstly, the strength of the relationship between potential risk factors and AR was evaluated using logistic regression analysis. Secondly, the pre- and postnatal factors after adjustment for risk factors using logistic regression analyses were used.p<0.05.In Model 1, the independent risk factors for AR were adjusted for the child’s age and gender. Model 2 was further adjusted for socioeconomic factors: paternal and maternal education, household income, family size and family numbers and city. All statistical analysis used SPSS statistical software package for Windows version 17.0.1 . Significance was defined as a Of the 21,030 children from the six grades of the chosen schools, 20,803 (response rate: 99.0%) participated in the survey and returned completed questionnaires. Of the total sample, 49.6% were males and the mean age was 9.19 years (SD = 1.76). The vast majority  were from the Han ethnic group, with the remaining children  from ethnic minorities. The overall prevalence of AR was 9.8% in this population.p = 0.001).The prevalence rates of AR in children <7, 7, 8, 9, 10, 11, and ≥ 12 years were 9.1, 8.3, 10.2, 10.1, 10.1, 11.0 and 10.7%, respectively. There were statistically significant differences in the prevalence of AR among the different ages , post-term birth or delivery by cesarean section , and mother exhibiting depressive symptoms during the pre- and postnatal periods . However, parental smoking, and/or parental alcohol abuse had no significant association with AR measurements.As shown in p<0.001), post-term birth and AR , cesarean section and AR , remained statistically significant after adjusting for all confounders. Of the aforementioned pre- and postnatal risk factors, the other significant risk factor for AR was the mother exhibiting depressive symptoms during the pre- and postnatal periods . In addition, interactions between the covariates in multivariate models showed no significant differences.In multivariate logistic regression for Model 1 , after aThe national pattern of prevalence and associated pre- and postnatal risk factors of childhood allergies in China is available in this study. The overall prevalence of AR in Chinese school-aged children according to this study was 9.6%, and significantly higher rates of AR were observed in boys than in girls . However, the prevalence of AR exhibited a different pattern from those of previously reported studies, peaking at the age of 11(11.0%), and not in younger school children . The mosThe same conclusions were drawn in a multidisciplinary review regarding the effect of breastfeeding on later development of allergic diseases . The bioIn general, some authors argue that low gestational age is associated with a decreased risk of AR . HoweverA recent WHO survey revealed that China had the world’s highest rate of cesarean sections (up to 46.2%), of which 25% were done without medical indications, far exceeding the upper threshold of 15% recommended by WHO . SimilarAnother new finding in this study was the positive relationship between depressive symptoms in mothers during the pre- and postnatal periods and subsequent AR in the school-age offspring. However, it is unclear whether there are any critical periods of exposure. The association of AR with pre- and postnatal exposure to maternal depressive symptoms has not been reported in human studies, but some explanation has been provided. Maternal stress associated with sustained excessive cortisol secretion could affect the developing immune system, especially Th1/Th2 cell balance of the child . AlterinWe acknowledge that there are some limitations to our study. Firstly, there was a lack of a more-expanded panel of questions regarding family history of atopic diseases because this study was originally designed to address the issue of childhood sleep patterns. In a published meta-analysis study , data shThe most important strength of this study was its ability to examine the association between pre-, postnatal risk factors and AR in a large cross-sectional survey with high representation. To our knowledge, this is the first report to provide information about breastfeeding, age of gestation, mode of delivery and mother exhibiting signs of depression during the pre- and postnatal periods between risk factors and AR in Chinese school-aged children.In this study, we provide evidence of how the pre- and postnatal risk factors such as post-term birth, cesarean section, no exclusive breastfeeding in the first 4 months, and the mother exhibiting depressive symptoms during the pre- and postnatal periods, might be related to the prevalence of AR in children. There is a need for worldwide research to further explore these factors and to develop policies for early intervention in a child’s life to prevent AR. Meanwhile, additional research will most likely ensue to confirm the critical exposure period and the underlying immunologic mechanisms.


Niphates digitalis marine sponge. Here we begin to characterize the mechanism of niphatenones in blocking AR transcriptional activity. Both enantiomers had similar IC50 values of 6 µM for inhibiting the full-length AR in a functional transcriptional assay. However, (S)-niphatenone had significantly better activity against the AR NTD compared to (R)-niphatenone. Consistent with niphatenones binding to and inhibiting transactivation of AR NTD, niphatenones inhibited AR splice variant. Niphatenone did not affect the transcriptional activity of the related progesterone receptor, but slightly decreased glucocorticoid receptor (GR) activity and covalently bound to GR activation function-1 (AF-1) region. Niphatenone blocked N/C interactions of AR without altering either AR protein levels or its intracellular localization in response to androgen. Alkylation with glutathione suggests that niphatenones are not a feasible scaffold for further drug development.Androgen ablation therapy causes a temporary reduction in tumor burden in patients with advanced prostate cancer. Unfortunately the malignancy will return to form lethal castration-recurrent prostate cancer (CRPC). The androgen receptor (AR) remains transcriptionally active in CRPC in spite of castrate levels of androgens in the blood. AR transcriptional activity resides in its N-terminal domain (NTD). Possible mechanisms of continued AR transcriptional activity may include, at least in part, expression of constitutively active splice variants of AR that lack the C-terminal ligand-binding domain (LBD). Current therapies that target the AR LBD, would not be effective against these AR variants. Currently no drugs are clinically available that target the AR NTD which should be effective against these AR variants as well as full-length AR. Niphatenones were originally isolated and identified in active extracts from  Recurrence of prostate cancer after primary therapies occurs in approximately 20% of patients. These recurrent patients receive androgen ablation therapy that causes a temporary reduction in tumor burden, but the malignancy will eventually begin to grow again in the absence of testicular androgens to form castration-recurrent prostate cancer (CRPC). A rising titer of serum prostate-specific antigen (PSA) signifies biochemical failure and precedes clinical symptoms of the emergence of lethal CRPC. PSA is an example of a gene that is transcriptionally regulated by androgen receptor (AR). Thus there is continued transactivation of AR even though blood levels of androgen are low.Androgens mediate their effects through the AR which is a ligand-activated transcription factor. This receptor contains several functional domains that include: the ligand-binding domain (LBD) to which androgens and antiandrogens bind; the hinge region which contains a nuclear translocation sequence; the DNA-binding domain (DBD) which binds to sequences called androgen response elements (AREs) in the enhancers and promoters of target genes; and the N-terminal domain (NTD) which contains activation function-1 (AF-1) which is responsible for most of the AR's transcriptional activity. The NTD is not a folded domain but rather intrinsically disordered or in a pre-molten globular structure In the absence of androgen, AR is complexed with chaperone proteins and located in the cytoplasm. Upon binding ligand, the receptor becomes hyperphosphoryated, translocates to the nucleus, dimerizes in an antiparallel orientation through N/C  interactions, and interacts with other co-regulatory proteins including bridging factors and the basal transcriptional machinery on AREs of target genes to initiate transcription. AR regulates genes involved in proliferation and survival of prostate cancer cells and is a validated drug target for all stages of prostate cancer.Current therapies directed at AR including androgen ablation , and antiandrogens  all target the AR LBD and will eventually fail Niphates digitalis that represents a novel structural class of AR antagonist. Niphatenone B binds covalently to the AF-1 region of the AR NTD and blocks the proliferation of prostate cancer cells that are dependent on functional AR Niphatenone B is a glycerol ether initially isolated from the marine sponge S)-niphatenone B or (R)-niphatenone B were synthesized with greater than 98% enantiometrically purity as previously reported -luciferase, probasin (PB)-luciferase, PRE-luciferase, GRE-Luciferase, 5xGal4UAS-TATA-luciferase and AR-(1-558)-Gal4 DBD, AR-YFP and ARAll experiments were carried out at concentration of 7.8 µM niphatenone, 25 µM EPI-002, 1 nM R1881, and 10 µM antiandrogens  except where specifically indicated.S)-niphatenone B or (R)-niphatenone B before addition of R1881 under serum-free and phenol red-free conditions. After 48 hours of exposure, cells were harvested, luciferase activity measured and normalized to protein concentration. IC50 values were calculated using OriginPro 8.1 Software .LNCaP cells were transfected with the PB-luciferase and PSA(6.1 kb)-luciferase reporters and pre-treated the next day for 1 hour with serial dilutions of -Gal4 DBD prior pre-treatment with 7.0 µM niphatenone B enantiomers or EPI-002, a positive control which binds to the AR NTD to block AR transcriptional activity var567es encoding constitutively active AR splice variant that lacks the LBD was co-transfected with PB-luciferase reporter in the AR-negative Cos-1 cell line. Six hours post-transfection, cells were treated with 1 µM each niphatenone stereoisomer and incubated for another 24 hours. Luciferase activity was measured and normalized to protein concentration determined by the Bradford assay.The expression plasmid ARS)-niphatenone B or with bicalutamide for 1 hour before addition of R1881, or progesterone , or dexamethasone. After 48-hours of treatment, cells were lyzed and analyzed for luciferase activities that were normalized to protein concentrations.LNCaP cells were transfected with plasmids encoding PSA(6.1 kb)-luciferase, a progesterone receptor (PR)-beta expression vector together with PRE-luciferase or a glucocorticoid receptor (GR)-expression vector and GRE-Luciferase reporter in serum-free, phenol red-free medium. Twenty-four hours later, the cells were pre-treated with  Kits  were employed according to the manufacture's protocols. Serial dilution was performed for each small molecule, and solvent was compensated to ensure equal volume (v/v %) of DMSO and ethanol in each sample. Assays were done in technical triplicates using Greiner 384 black clear-bottom plates. Fluorescence polarization was measured by Infinite M1000  with excitation wavelength of 470 nm and emission wavelength of 535 nm.S)-niphatenone B before incubation with R1881 for additional 24 hours. Luciferase activity was measured and normalized to protein.The mammalian two-hybrid N/C interaction assay was performed using CV1 cells that were transfected with GAL4DBD-AR LBD-(AR628-919) encoding the wild type AR LBD C-terminus amino acid residues 628-919 fused to the Gal4 DBD, VP16-AR-(1-565) that encodes the VP16 transactivation domain fused to amino residues 1–565 of AR NTD and Gal4-luciferase reporter. Transfected cells were pre-treated for 1 hour with bicalutamide or (S)-niphatenone B or bicalutamide prior to addition of R1881 for 16 hours under serum- and phenol red-free conditions. Total RNA was isolated using RNeasy mini kit (Qiagen) and transcripts for KLK2 and KLK3/PSA measured by quantitative real-time (qRT)-PCR in triplicates for each biological sample. Expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene. Primers employed were previously described LNCaP cells were serum starved for 48 hours. Cells were treated for 1 hour with vehicle, (S)-niphatenone B or bicalutamide for 1 hour prior to R1881 addition for 16 hours. Cells were harvested and whole-cell lysates were subjected to 10% SDS-PAGE. Membranes were probed with anti-AR PG21 antibody and normalized to protein levels of β-Actin.LNCaP cells were serum starved for 48 hours then treated with vehicle, (S)-niphatenone B, bicalutamide or enzalutamide (MDV-3100) for 1 hour prior to R1881 exposure for 2 hours. Cells were fixed with paraformaldehyde, stained with DAPI, and examined by fluorescence microscopy .LNCaP cells seeded on sterile cover slips were transfected with the expression vector for AR-YFP fusion protein. Following 24 hours incubation, cells were treated with vehicle,  or GR AF-1 (amino acid residues 77–262) were carried out by mixing 10 µM AR AF-1 or GR AF-1 with 20 µM niphatenone B 44 or EPI-054 probes containing an alkyne group or with DMSO in equivalent amounts. Binding reaction was incubated on ice for 45 minutes. Niphatenone B or EPI probe were then labeled with fluorescein azide by Click chemistry at room temperature for 30 minutes. Proteins were separated from free probes on reducing SDS-PAGE and fluorescein was detected by an image analyzer. Coomassie blue staining was done on the same gels to show equal loading of AF-1 protein to each lane.2O were added and the mixture was warmed to 37°C. The mixture was monitored by NMR every few hours .HEPES, TCEP, reference material pentachlorobenzene, and niphatenone B enantiomers (0.014 mmol) or EPI-002 (0.014 mmol), were added separately to a small vial. After deuterated DMSO was added, the sample was run NMR (0 h). Glutathione and Dt test. Analysis was performed using GraphPad Prism .Values are presented as mean ± SD or ± SEM. Comparison of two populations was made by use of Student's Inhibitors of the AR NTD would be beneficial for blocking all AR species because the NTD contains the AF-1 region that is essential for AR transcriptional activity S)-Niphatenone B IC50 was 5.7±0.6 µM and (R)-niphatenone B IC50 was 6.3±0.5 µM. To determine whether the DNA recognition sequence (ARE and/or promoter/enhancer sequence) influenced enantiomer IC50 values, a second AR-driven reporter was tested. IC50 values using the PSA(6.1 kb)-luciferase reporter were 5.17±0.59 µM and 6.25±0.82 µM for (S)-Niphatenone B and (R)-niphatenone B respectively. Thus niphatenone B enantiomers block androgen induction of AR-driven reporters thereby supporting that they may be a new class of AR antagonist.To determine IC50 values for inhibition of AR activity by each niphatenone enantiomer, the AR-driven PB-luciferase reporter was employed that contains the androgen responsive region with two AREs. Both enantiomers had IC50 values in the range of 6 µM using this AR-driven reporter. (S) configuration was significantly better than the (R) configuration  configuration showed significantly better inhibition of transactivation of the AR NTD (S)-Niphatenone B for activity against the AR, the ability of (S)-niphatenone B to inhibit the steroidal transcriptional activity of full-length AR, and structurally related PR-β and GR were compared in LNCaP cells. As expected, the clinically used antiandrogen, bicalutamide (which binds the LBDs of both AR and PR) completely blocked androgen-induced AR (S)-niphatenone B inhibited androgen-induced AR-driven PSA-luciferase activity by approximately 80% (p<0.0001), but did not inhibit PR transcriptional activity at this same concentration. (S)-niphatenone B had a modest but statistically significant effect on GR-driven transcriptional activity -nipactivity . No tranactivity . Thus thS)-niphatenone B interferes with ligand-binding, we evaluated its ability to compete for the LBD of AR and related steroid receptors, PR-β and GR. At concentrations used to block the AR activity, (S)-niphatenone B did not interfere with ligand-binding to the AR, PR and GR LBDs as shown in fluorescence polarization assays (S)-niphatenone B interfered with ligand-binding. Such a result may indicate off-target effects at high concentrations.To test whether (n assays . HoweverS)-niphatenone B works by a similar mechanism to EPI-001, the mammalian two-hybrid system was used to measure N/C interaction in CV-1 cells that do not express endogenous AR. In the absence of androgen, no N/C interaction was detected and as expected, in its presence, androgen induced robust interaction (S)-niphatenone B .EPI-001 binds the AF-1 region in the AR NTD which blocks AR N/C interaction eraction . BicalutPSA and KLK2 genes are dependent on functional AR. These genes contain bone fide AREs to which the AR binds to initiate transcription. To test whether niphatenone B has an effect on endogenous gene expression, the levels of PSA and KLK2 transcripts were measured in LNCaP cells in response to androgen with and without (S)-niphatenone B treatment. Androgen induced approximately a 9-fold increase in PSA mRNA levels but this induction was inhibited by approximately 70% by (S)-niphatenone B (S)-niphatenone B also blocked androgen-induction of KLK2 gene expression (Androgen induction of expression of tenone B . This ispression . TogetheS)-niphatenone B in the presence or absence of androgen. In the absence of androgen, there was no significant change of the levels of AR protein in LNCaP cells with (S)-niphatenone B (p = 0.03) decreased levels of AR protein compared to androgen only (lane 2).The antagonist effect of niphatenones on AR activity may be mediated by alterations of levels or subcellular localization of AR protein. To begin to further reveal the molecular mechanism of action of niphatenones, LNCaP cells were exposed to -niphatenone B revealed covalent binding to the AR AF-1 peptide in a cell-free assay Previously, Click chemistryusing a propargyl ether of -niphatenone inhibited transactivation of the AR NTD better than (R)-niphatenone; 4) (S)-niphatenone had no effect on PR activity; 5) niphatenone B did not affect the levels or intracellular localization of AR protein; 6) at higher concentrations both enantiomers had off-target effects that included blocking androgen binding to the AR LBD, forming adducts with glutathione, and covalent binding to recombinant GR AF-1 peptide.Here niphatenone B enantiomers were characterized and revealed: 1) comparable IC50 values of approximately 6 µM for blocking full-length AR activity; 2) both enantiomers inhibited constitutively active AR splice variant v567es; 3)  and is correlated to poor survival for patients with CRPC S)-niphatenone B had no effect of PR transcriptional activity while had a modest effect (∼20%) on GR and substantially attenuated AR (>80%) transcriptional activity. However, niphatenones at concentrations above 10 µM lacked desirable specificity and have off-target binding. The first indication of potential off-targets came from the ligand-binding assay. At a concentration of 30 µM, niphatenone reduced polarization that is caused by decreased binding of fluorescently labeled hormone (fluoromone) to the LBDs of AR, PR and GR. This result suggests binding of niphatenone B to the AR-, PR- and GR-LBDs in addition to its covalent binding to AR NTD. The NTD and LBD of AR lack sequence similarities. The Click chemistry probe for niphatenone B at 20 µM bound covalently to both the AR AF-1 and GR AF-1 proteins that have limited similar amino acid residue sequence - and (R)-niphatenone B or DMSO vehicle control for 24 h. Cells were labeled and BrdU incorporation was measured by ELISA. Data is presented as percentage of vehicle control. Bars represent the mean ± SEM of three independent experiments.(EPS)Click here for additional data file.Figure S2LNCaP cells require forced expression of PR-β or GR to measure transcriptional activity in response to cognate ligands. LNCaP cells co-transfected with empty vector or expression vector for PR-β. (A) or GR (B) and the corresponding luciferase reporter (PRE-Luc or GRE-Luc), under serum-free and phenol-red free conditions, were exposed to 10 nM of progesterone, dexamethasone or ethanol vehicle control for 48 h. Representative figures of three independent experiments. Bars represent the mean ± SD of three technical replicates.(EPS)Click here for additional data file.Figure S3AR NTD inhibitor inhibits all AR species. Androgen receptor (AR) transcriptional activity can be blocked by targeting either the LBD or the NTD. The first approach is the basis for development of abiraterone acetate and anti-androgens (AA) used in the clinic to treat prostate cancer. Abiraterone acetate blocks the synthesis of androgen. Both abiraterone acetate and antiandrogens that target the LBD have transient therapeutic effects. Eventually the cancer will become resistant to inhibitors of AR LBD possibly via expression of AR splice variants. On the other hand, by targeting the NTD which possesses most if not all the transcriptional activity of AR, an NTD inhibitor (NTDI) will effectively inhibit transcriptional activity of full-length AR, regardless of ligand binding, and truncated constitutively active AR splice variant lacking LBD.(EPS)Click here for additional data file.


The results show that the optimum NaOH molarity found in this study is 12 M due to the highest strength  of 15.79% with lower water absorption and specific gravity. The optimum LUSI mud/Alkaline activator (LM/AA) ratio of 1.7 and the Na2SiO3/NaOH ratio of 0.4 gives the highest strength with AIV value of 15.42% with specific gravity of 1.10 g/cm3 and water absorption of 4.7%. The major synthesized crystalline phases were identified as sodalite, quartz and albite. Scanning Electron Microscope (SEM) image showed more complete geopolymer matrix which contributes to highest strength of ALGA produced.This paper presents the mechanical function and characterization of an artificial lightweight geopolymer aggregate (ALGA) using LUSI (Sidoarjo mud) and alkaline activator as source materials. LUSI stands for LU-Lumpur and SI-Sidoarjo, meaning mud from Sidoarjo which erupted near the Banjarpanji-1 exploration well in Sidoarjo, East Java, Indonesia on 27 May 2006. The effect of NaOH molarity, LUSI mud/Alkaline activator (LM/AA) ratio, and Na The high volume of LUSI  mud that began erupting near the Banjarpanji-1 exploration well in Sidoarjo, East Java, Indonesia needed to be converted into a useful and valuable alternative resource. This eruption affected an area of almost three square miles to a depth of 65 feet and thirty thousand people were displaced, costing Indonesia 3.7 billion dollars in damages and damage control. The total cost of damage and damage control reached 488 million US dollars (USD) in 2011 and increased to 730.7 million USD in 2014 [et al. [et al. [During the last decade, considerable research efforts have been directed towards the development of inorganic geopolymers, due to the wide range of potential applications for these materials. Geopolymerization is an innovative technology that can transform several aluminosilicate materials into useful products called geopolymers or inorganic polymers ,9. The get al. , and Har [et al. . There i2SiO3/NaOH ratio to the mechanical function and characterization of ALGA produced.Most countries worldwide have taken into account the impact of earthquake forces in designing the lightweight structure ,13. Ligh3. After 8 M, the specific gravity of ALGA is higher with increasing NaOH molarity. However, high specific gravity was also detected at 6 M NaOH molarity due to insufficient concentration of NaOH to be reacted with LUSI mud particles. The specific gravity of ALGA at 8, 10, and 12 M are merely different with 1.12 g/cm3, 1.14 and 1.18 g/cm3, respectively. The high specific gravity is detected at 14 and 16 M due to the exceeded concentration of NaOH.The specific gravity of ALGA produced at various NaOH molarity is shown in 2SiO3/NaOH ratio are shown in 2SiO3/NaOH ratio of 0.4 showed decreases of specific gravity, then kept increasing after 0.4 for all types of LM/AA ratio. The lowest specific gravity is observed at LM/AA ratio of 1.7 and Na2SiO3/NaOH ratio of 0.4 with 1.10 g/cm3. This value is merely different with ALGA produced at Na2SiO3/NaOH ratio of 0.2 and 0.6 with 1.13 and 1.12 g/cm3, respectively. For LM/AA ratio of 1.8 and 1.9, insignificant results were observed due to high specific gravity produced.The specific gravity of ALGA produced at various LUSI mud/Alkaline activator (LM/AA) ratio and Na2SiO3/NaOH ratio. There is no specific pattern for water absorption produced. The highest water absorption is found at LM/AA ratio of 1.9 and Na2SiO3/NaOH ratio of 0.8 with 6.8% and the lowest water absorption is found at LM/AA ratio of 1.7 and Na2SiO3/NaOH ratio of 0.4 with 4.7%.+ ions to allow for complete polymerization of the network [3+ and Si4+ ions released from the alumina-silicates to participate in geopolymerization process with the optimum alkalinity medium. Sufficient alkali must be present for the complete dissolution process, then giving an increased geopolymerisation rate and higher strength [ network . The besstrength . The AIVstrength  as excesstrength .2SiO3/NaOH ratio. The lowest AIV was found at the LM/AA ratio of 1.7 and Na2SiO3/NaOH ratio of 0.4 with 15.42%. The highest AIV is observed at LM/AA ratio of 1.9 and Na2SiO3/NaOH ratio of 1.0 with 27.40%, indicating lower strength. High LM/AA ratio indicates low workability and this caused difficulty during the palletizing process where failure in providing good compaction may reduce the strength of the ALGA produced. This statement also proved by Kong et al. [2SiO3/NaOH ratio of 0.2 then decreased at a 0.4 ratio. After that, the AIV value keeps increasing at a Na2SiO3/NaOH ratio of 0.6, 0.8 and 1.0 for all LM/AA ratio samples. For low Na2SiO3/NaOH ratio of 0.2, the excess of Na+ ions was believed to exist in structure, which will form the sodium carbonate, then affect the geopolymerization process and resulted in lower strength. For high Na2SiO3/NaOH ratio, excess of Na2SiO3 may hinder the evaporation of water and structure formation [2SiO3/NaOH ratio of 0.4 is believed to provide the optimum rate of geopolymerization process due to the highest strength  with lower water absorption and specific gravity. The correct proportion and spatial distribution of an aluminosilicate network will maximize the mechanical strength of the geopolymer matrix [g et al.  which coormation . The besr matrix .4Al3Si3O12Cl), albite (NaAlSi3O8) and quartz. The broad humps were detected at 2θ = 21°–36°, suggesting that amorphous phases are present showing the geopolymerization was occurred. High intensity has been detected at 2θ = 22.2° at 6 M indicating quartz, showing it did not take part in geopolymerization process. Then the high intensities observed at 2θ = 24.6° at others NaOH molarity which are all associated to the new phase of sodalite (S) formed. However the highest intensity peak of 12 M was found at 2θ = 35.8° which indicating the high formation of albite (Al) peak appeared. According to Eun Oh et al. [XRD analysis of ALGA from LUSI mud at various NaOH molarity are shown in h et al. ,21, the 3KAl4Si4O16) peaks has were detected at 2θ = 31° at 6 and 8 M, then shifted to the left at 2θ = 30.0° at 10, 12, 14 and 16 M. The anorthite (CaAl2Si2O8) phase also detected at 2θ = 23.0° at all molarities, except at 6 M. The presence of anorthite phase indicates the reaction between calcium from the LUSI mud with alumina-silicate. Exception of anorthite at 6 M was due to low concentration of NaOH to be reacted with calcium in LUSI mud.The nepheline  of Na4Al3Si3O12Cl, quartz (Q), albite (Al), anorthite (An) and nepheline (N) that strengthen the ALGA produced [2SiO3/NaOH ratio of 0.4, the major phases were sodalite (S), sodium aluminium silicate (SAS), anorthite (An) and albite (Al).Meanwhile, the XRD analysis of ALGA produced from LUSI mud at the best three mix design LM/AA ratio and Naproduced . The bro2SiO3/NaOH ratio of 0.4, the major phases were sodalite (S), sodium aluminium silicate (SAS), anorthite (An), and albite (Al). The crystalline phase of sodalite also has been found by Criado et al. [et al. [For LM/AA ratio of 1.9 and Nao et al.  and Alva [et al.  for fly  [et al. , thus coThe alkaline activation of LUSI mud caused the dissolution of silicate and aluminate species into solution. The dissolved alumina (Al) will firstly react with the silicate (from sodium silicate) to form the silicate oligomers, then grows and begins to crystallize forming zeolites .et al. [The presence of efflorescence was observed on the samples ALGA of 16 M f. Accordet al. , the effet al. .ALGA with NaOH molarity 14 and 16 M showed the more dense structure due to excessive concentration of NaOH in geopolymer matrix. These contribute to the high density of ALGA produced, but low strength due to the presence of efflorescence.2SiO3/NaOH ratio and control samples  are shown in 2SiO3/NaOH ratio of 0.4 (3).The SEM micrographs of ALGA produced at the best three mix design (high strength) of LM/AA ratio with Nao of 0.4 a showed 2SiO3/NaOH ratio of 0.4  spectra of ALGA produced at various NaOH molarity. Transformation took place during synthesis of geopolymer was indicated by the different absorption frequencies of ALGA produced. The O–C–O stretching vibration (band 4) exists in the ALGA produced at 12, 14, and 16 M at 1431–1450 cm−1, but is absent at low molarity of 6, 8, and 10 M. The asymmetric stretching vibrations of Si-O-Si and Al-O-Si at 1012 cm−1 (band 5) for 6, 8, and 10 M are the same, but then shifted to the lower frequency of 1007 cm−1 at 12 M, then turn to 1011 cm−1 at 14 and 16 M. This indicates the formation of a new product of sodium aluminum silicate (SAS) phase as proved by XRD results, which is associated with the higher dissolution of LUSI mud in the strong molarity of alkaline activator solution .−1) assigned to symmetric stretching vibrations of Si–O–Si and Al–O–Si [−1 appeared at all ALGA produced at 6–16M, which assigned to bending vibrations of Si–O–Si and O–Si–O bonds [The Al–O–Si stretching vibrations (band 6) appeared in all samples at 6–16 M. Moreover, the structural reorganization of LUSI mud is evidenced by the appearance of band 7 at lower frequencies . The bands 1, 2, and 3 at 1627–1630 and 2921–3451 cm−1 arise from the presence of structural water remaining in the geopolymer matrix of ALGA and control sample. The most characteristic difference observed between the FTIR spectrum of ALGA and control sample is the band 5 of asymmetric stretching vibrations of Si–O–Si and Al–O–Si. The band of 1075 cm−1 in the FTIR spectrum of aggregate produced without alkaline activator (control samples), then shifts to lower frequencies (995–1012 cm−1) in the FTIR spectra of ALGA produced at LM/AA ratio of 1.7, 1.8 and 1.9 with Na2SiO3/NaOH ratio of 0.4. Verdolotti et al. [−1 showed the variability of the bond angles and bond lengths of the tetrahedral structures around the silicon atoms.i et al.  stated t−1 (band 6) that appeared at ALGA produced the best mix designs which were assigned to stretching vibration of Al–O–Si bonds [The absorbance peak at 731 cmSi bonds ,31, but 2 + Al2O3 + Fe2O3 >70% indicated that this LUSI mud can be used as pozzolan materials (ASTM C-618) [The present study was conducted on the LUSI mud collected from the eruption sites near Sidoarjo, East Java, Indonesia. The received batches of LUSI mud were already partially dried. The LUSI mud needs to be treated  before transforming into the ash form to simplify the palletizing process of aggregate and remove any other coarser particles. The chemical composition of LUSI mud is done by X-ray Flourescence (XRF) and the details are shown in M C-618)  and is s2/g. Finer particles provide high surface area to react in geopolymerization process and affect the strength of geopolymer. LUSI mud needs to be in fine particles to simplify the mixing process of geopolymer mixture in the production of ALGA. The mean particle size of the LUSI mud is dominated by particles in the size d (0.5) of 121 µm with specific surface area of 0.151 m2). This material exhibits its highest peak at 2θ = 26.6° due to higher intensity of quartz (Q) (SiO2). This was also demonstrated by the XRF results which shows that the higher contents of SiO2 represented with high peak of intensity in XRD results. LUSI mud showed two other intense diffraction peaks at 2θ = 21.0°, 35.0°, 36.6°, 39.2° and 50.2°, which are associated with quartz (Q). The 2θ values of 27.9° revealed the mullite and 31.8° revealed the hematite (Fe2O3) element. The moganite (M) and crystobalite (C) were also detected at 2θ = 19.7° and 21.8°, respectively.An X-ray diffraction (XRD) analysis of LUSI mud is shown in LUSI mud has a plate-like structure which was similar to kaolin as shown in −1, corresponding to the OH- stretching vibration. The OH– and H–O–H stretching vibration are assigned at 2924 and 2854 cm−1. The H–O–H bending at 1638 cm−1 assigned to the weakly-bound water molecules, which are adsorbed on the surface or trapped in the large cavities between the agglomerated LUSI mud. The stretching vibration of O–C–O is assigned at 1424 cm−1. Band at 1034 cm−1 was assigned to Si-O bonds in the SiO4 molecules. The bands at 797 and 778 cm−1 are assigned to Si–O–Si symmetric stretching. The band at 695 cm−1 is assigned to Al–O–Si bending, meanwhile the band at 467 cm−1 is assigned to Si–O–Si and O–Si–O bending.2, 9.4% Na2O and 60.5% H2O (modulus SiO2/Na2O = 3.2), specific gravity at 20 °C = 1.4 g/cm3 and viscosity at 20 °C = 0.4 Pa∙s.Sodium hydroxide (NaOH) used in this study was from the pallet type of Formosoda-P from Formosa Plastic Corporation, Taiwan. Sodium silicate used was obtained from South Pacific Chemical Industries Sdn. Bhd. (SPCI), Malaysia. Sodium silicate has a chemical composition of 30.1% SiOIn order to find the effect of NaOH molarity to the ALGA, the ratio of LUSI mud/Alkaline activator (LM/AA) also should be fixed to 1.7 for all mixtures. The mass of LUSI mud and alkaline activator solution were kept constant for all 6, 8, 10, 12, 14 and 16 M ,35 with 2SiO3/NaOH are shown in After finding the best NaOH molarity, the optimum mix design on producing ALGA should be studied. The details of the mix design of various ratios of LM/AA and Na2SiO3/NaOH used are 0.2, 0.4, 0.6, 0.8, and 1.0. The best mix design ratios then compared with the control sample .In this experiment, the ratio of LM/AA used were 1.7, 1.8 and 1.9. These values were chosen due to the workability during the palletizing process to form the aggregate. The workability is too high if LM/AA below 1.7 is used, meanwhile the workability is too low if LM/AA above than 1.9 is used. The ratio of Na2SiO3 and NaOH solution) needs to be prepared for 24 h prior to use. After all materials have been prepared and weighted, the geopolymer paste is prepared by mixing LUSI mud that has been treated to ash form with the alkaline activator for 10 min by using a hand mixer, forming a homogeneous paste.Alkaline activator solution .The water absorption test has been conducted according to ASTM C140  to obtaiAggregate impact value (AIV) test was done to determine the impact value of artificial geopolymer aggregate from LUSI mud as per MS30:PART 10:1995. The value of AIV represents the strength of aggregate produced.X-ray diffraction (XRD) patterns were obtained by using X-ray diffractometer XRD-6000  using Cu-Kα radiation generated at 30 Ma and 40 kV. The XRD test should be carried out to get the phase analysis of ALGA produced.Morphology of LUSI mud for ALGA produced were revealed by using JSM-6460LA model Scanning Electron Microscope (JEOL) . In this testing, the samples were cut into fine and smooth small pieces portion. Then, all specimens were coated with palladium by using Auto Fine Coater JEOL JFC 1600 model before the testing.−1 with resolution of 4 cm−1.Fourier transform infrared (FTIR) spectroscopy analysis of ALGA of different parameters are performed by using Perkin Elmer FTIR Spectrum RX1 Spectrometer. The samples are prepared in powder form and KBr pellet technique. The samples were scanned from 500 to 4000 cm3O8) and sodium aluminium silicate (SAS) appeared at ALGA produced at 12 M which might affect the highest strength produced.The optimum NaOH molarity found in this study is 12 M due to the highest strength  of 15.79% with lower water absorption and specific gravity to be performed on ALGA lightweight concrete. The continuous gel-like area with some voids at 12 M is made of pure geopolymer binder which strengthens the structure itself and contributes to high strength. The new phases of albite (Al) , silicon dioxide (SiO2) and quartz (Q). SEM image showed more complete geopolymer matrix which contributes to highest strength of ALGA produced. The band of 1075 cm−1 assigned to asymmetric stretching vibrations of Si–O–Si and Al–O–Si in the FTIR spectrum of control sample shifts to lower frequencies (~995–1012 cm−1) in the FTIR spectra of ALGA produced at the best mix design. The broadness of the absorbance band at 820–1250 cm−1 showed the variability of the bond angles and bond lengths of the tetrahedral structures around the silicon atoms.For selection of optimum mix design, the LUSI mud/Alkaline activator (LM/AA) ratio of 1.7 and NaThe performance of ALGA can be further analyzed in lightweight concrete application for the future works.


Background: Arsenic as an environmental toxicant is able to exert malformations in male reproductive system by inducing oxidative stress. Vitamin E (Vit.E) is known as antioxidant vitamin.Objective: The aim of this study was to investigate the harmful effects of sodium arsenite on sperm parameters and the antioxidant effects of Vit.E on sperm anomalies in sodium arsenite treated rats.Materials and Methods: Adult male rats were divided into 4 groups: control, sodium arsenite (8 mg/kg/day), Vit.E (100 mg/kg/day) and sodium arsenite+Vit.E. Oral treatments were performed till 8 weeks. Body and left testis weight were recorded and then left caudal epididymis was cut in Ham's F10. Released spermatozoa were used to analyze number, motility, viability and abnormalities of the sperm. Sperm chromatin quality was assessed by nuclear staining using acridine orange and aniline blue.Results: Body and testis weight showed no significant change in 4 groups (p>0.05). A significant decrease in the number, motility, viability and normal sperm morphology was found in sodium arsenite-treated rats compared to the control (p<0.001). Sodium arsenite had no effect on sperm DNA integrity and histon-protamine replacement (p>0.05). In sodium arsenite+Vit.E group, Vit.E could significantly compensate the harmful effects of sodium arsenite on sperm number, motility, viability and morphology compared to sodium arsenite group. In addition, sperm viability and motility was significantly increased in rats treated with Vit.E alone compared to the control and sodium arsenite+Vit.E group.Conclusion: Vitamin E could compensate the adverse effects of sodium arsenite on sperm parameters in adult rats. Arsenic, a sulfhydryl-reactive metalloid, is a major environmental toxicant produced by the burning of arsenic contaminated coal and glass manufacturing . In drinArsenic is a known human carcinogen  and is aArsenic can also induce male reproductive toxicity such as dose-dependent decrease in testes and accessory sex organ weights . It may The integrity of sperm DNA is an important factor for the success of fertilization as well as normal development of the embryo, fetus and child . SeveralIt is now documented that vitamin E (Vit.E), as a potent antioxidant , protectAnimals and treatmentsoC and fed with standard commercial laboratory chew and water. This experimental study was performed on adult albino male Wistar rats (250±20 gr). The animals were purchased from Pasture's Institute, Iran. The animals were housed in plastic cages at 12-h light/dark cycle, 24±2distilled water, sodium arsenite , Vit.E  and sodium arsenite +Vit.E. The reagents were orally given to the rats by gavage for 8 weeks “as the duration of spermatogenesis in wistar rats is 52 days” (Adult rats were divided into four groups (n=6 for each group): control which received 52 days” . At the end of the treatments, the animals were weighed, anesthetized by the injection of pentobarbital (60 mg/kg) and sacrificed. Left testis and cauda epididymis of the animals were dissected. The testis was cleared from fat tissue and its weight was recorded.Sperm countThe dissected epididymis of each animal was transferred into 10 ml Ham's F10 medium and cut to small slices, in order to swim out the sperm into the medium. After 10 min of diffusion, 1 ml of the solution was diluted with 9 ml formaldehyde fixative. The diluted solution was transferred into each chamber of Neubauer hemocytometer and sperm heads was manually counted under a microscope. Sperm count was performed according to WHO guidelines  and dataSperm motilityAssessment of sperm motility was done according to WHO protocol . In brieSperm viabilityEosin-nigrosin staining was used to asses sperm viability according to WHO protocol  Briefly,Sperm morphologyThe eosin-nigrosin stained slides were used to evaluate sperm morphology. One hundred sperm were observed to detect sperm abnormalities in each sample.Sperm chromatin qualityoC and stained with AO solution  for 10 min. The slides were gently washed by distilled water for 5 minutes and air dried. The stained smears were then observed under fluorescence microscope at 1000X magnification. Three types of staining patterns were considered in sperm head; green spermatozoa (double-stranded DNA), yellow and red spermatozoa (single-stranded DNA). At least 100 spermatozoa per slide were count to evaluate the percentage of double-stranded DNA in the spermatozoa. Thin smears were prepared from the sperm solution and allowed to air-dry. To test sperm DNA integrity, the smears were stained with acridine orange (AO). AO staining was performed according to a protocol described by Tejada and co-workers . In brieTo test sperm histon-protamine replacement, the smears were stained with aniline blue (AB). AB staining was carried out based on the method described by Wongand co-workers . In brieStatistical analysisResults are expressed as mean±SD for six animals per group. One-way analysis of variance (ANOVA) was used to assess the statistical significance of the data. p<0.05 was considered significant.Body and testis weightFor each animal, body and testis weight were recorded at the end of the treatments. There was no significant difference in body and testis weight in the four groups .Sperm countResults showed a highly significant decrease (p<0.001) in epididymal sperm number in sodium arsenite group compared to the control . Sodium Sperm motilityThe treatment of animals with sodium arsenite significantly (p<0.001) decreased the percentage of PMS and increased (p<0.001) the percentage of NPMS and NMS compared to the control . Rats treated with sodium arsenite+Vit.E showed a significant increase (p<0.001) in the percentage of PMS and decrease (p<0.001) in the percentage of NPMS as well as NMS as compared with sodium arsenite group . AnimalsSperm viabilityThere was a significant decrease (p<0.001) between the percentage of viable sperm in sodium arsenite group compared to the control. A significant increase (p<0.05) was observed in sperm viability in rats exposed to sodium arsenite+ VE compared to sodium arsenite group . Rats trSperm morphological anomaliesRats treated with sodium arsenite showed a significant increase (p<0.001) in abnormal sperm. In sodium arsenite+Vit.E group, Vit.E could significantly (p<0.05) reverse sperm morphological anomalies as compared with sodium arsenite group . Some spSperm chromatin qualitySpermatozoa stained with AO showed that sodium arsenite had no significant effect on the sperm DNA integrity compared to the control . In addiThis study examined the adverse effect of sodium arsenite on epididymal sperm parameters in adult rats. In addition, Vit.E showed to reverse the toxic effect of sodium arsenite on these parameters.In present study no significant difference was observed in the body and testis weight in sodium arsenite-exposed rats. Our results were in agreement with previous findings regarding the effect of sodium arsenite on the body  and testIn agreement with pervious study , our resChromatin and flagellum in mammalian spermatozoa contain large amount of thiol rich protamine and sulfhydryl groups respectively which involve in the maintenance of sperm stability and motility . The decAn interesting finding in sperm viability and motility assay was that Vit.E alone increased these parameters compared to the control. This effective result of Vit.E might also be due to its antioxidant role. This vitamin plays an important protective role for preventing the production of lipid peroxides by scavenging free radicals which are toxic for biological membranes . TherefoOur results showed a significant increase in sperm morphological anomalies in rats treated with sodium arsenite. It is documented that ROS generation can induce abnormal sperm morphology . It is ton these parameters in epididymal sperm of adult rat. It is likely that the effect of sodium arsenite on these parameters has been dose and duration-dependent.AO and AB staining are methods for determining sperm DNA integrity (double strand DNA versus single strand DNA)  and histArsenic is a potent endocrine disruptor . TherefoOur results indicate that sodium arsenite has a negative influence on sperm number, sperm motility, sperm viability and normal sperm morphology but not on DNA integrity and histon-protamine replacement in adult rats exposed with this toxicant. In addition, Vit.E is able to compensate the adverse effects of sodium arsenite on sperm number, motility, viability and normal sperm morphological anomalies.


Growing evidence suggests the presence of non-coding cis-antisense RNAs that regulate gene expression via antisense interaction. Recent studies also indicate the role of transcriptional interference in regulating expression of neighboring genes due to traffic of RNA polymerases from adjacent promoter regions. Previous models investigate these mechanisms independently, however, little is understood about how cells utilize coupling of these mechanisms in advantageous ways that could also be used to design novel synthetic genetic devices. Here, we present a mathematical modeling framework for antisense transcription that combines the effects of both transcriptional interference and cis-antisense regulation. We demonstrate the tunability of transcriptional interference through various parameters, and that coupling of transcriptional interference with cis-antisense RNA interaction gives rise to hypersensitive switches in expression of both antisense genes. When implementing additional positive and negative feed-back loops from proteins encoded by these genes, the system response acquires a bistable behavior. Our model shows that combining these multiple-levels of regulation allows fine-tuning of system parameters to give rise to a highly tunable output, ranging from a simple-first order response to biologically complex higher-order response such as tunable bistable switch. We identify important parameters affecting the cellular switch response in order to provide the design principles for tunable gene expression using antisense transcription. This presents an important insight into functional role of antisense transcription and its importance towards design of synthetic biological switches.Antisense transcription has been extensively recognized as a regulatory mechanism for gene expression across all kingdoms of life. Despite the broad importance and extensive experimental determination of  Non-ccis-antisense RNA interaction; or between transcripts encoded from different genome regions, which are only partially complementary, known as trans-antisense interaction. Antisense RNAs (asRNAs) are ubiquitous in all three domains of life and known to regulate gene expression in various ways [AR can arise between RNA transcripts encoded in opposite strands of the same DNA region, which are perfectly complementary, known as ous ways –22. asRNous ways  or, lessous ways . mRNA deous ways ,25–27. Fous ways –30. Otheous ways ,31, inhious ways ,32.During antisense transcription an additional mechanism affecting expression of neighboring genes is known as TI and is defined as the suppressive effect of transcription from one promoter on an adjacent promoter . Presenccis-antisense transcription, relatively little is known about its role in controlling cellular switching responses. Furthermore, majority of prior mathematical models have focused either on TI [Despite the broad importance and extensive experimental determination of er on TI  or AR [4er on TI ,43 mechaer on TI  as it caX and Y, respectively. The model calculates the outcome of every single round of transcription by tracking binding and movement of RNAP along three different regions along the DNA: the pX promoter region from position -70 to -1, the intermediate overlapping region of length L, which spans between positions +1 and L, and promoter pY region from position L+1 to L+70 . Promoter bound RNAP (IC) is assumed to occupy a footprint (fp) of 70 bp. Both RNAP binding (τBX±0.05τBX and τBY±0.05τBY) and initiation times (τIX±0.05τIX and τIY±0.05τIY) are assumed to follow a Gaussian distribution to account for inherent biological noise [bla system in E. coli [IX<τBX and τIY<τBY), thus self-occlusion at both promoters is neglected [IX<τBX and τIY<τBY, time between two consecutive RNAP firing events coincides with the time between two consecutive RNAP binding events at the promoter. Therefore, the RNAP firing rates or frequencies fX and fY from promoters pX and pY respectively are solely given by the inverse of the corresponding RNAP binding time . We define relative strength α of promoters pX-pY, as the ratio of RNAP firing rates (α = fY/fX). α can vary between three states, α<1 when pX is more aggressive than pY promoter, α~1 when pX and pY have comparable strengths, and α>1 when pY is more aggressive than pX. RNAPs are assumed to be in excess, thus we consider no effect on transcription due to sequestering of transcriptional machinery [Transcriptional initiation follows the McClure model, which describes the multi-step transcription initiation process using two main steps –46. Firsectively  to form al noise ,48. Aver E. coli ,50 and b E. coli . Average E. coli . We assueglected . Since τachinery . The binth and hth position for every ith and jth round of transcription   from pX and pY respectively. The time taken by the EC to cover this distance is given by the summation of product of RNAP velocity at a particular location along the DNA n is the number of nucleotides covered by the center of the EC, and vm is the local velocity of the EC at the mth position.In case of successful RNAP firing, we assume elongation starts with the center of an EC RNAP discretely moving to the first position of overlapping DNA . Footprint (fp) of an EC is considered to be 35 bp . Movemenx and y of length >L+70 . Time taken to traverse an antisense promoter starts when front end of the EC reaches position L  and the back end of the EC leaves position L+70 . Upon occlusion , jth round of transcription from pY does not occur. Similarly, for ith round of transcription at pX, occlusion is considered to occur if jth EC fired from pY is traversing pX region (from -70 to -1) when ith RNAP tries to bind at pX , (For j0-fp/2), .th EC fired from pX front end reaches the end of the overlapping region, i.e. position L, and encounters an IC stationed at pY , also known as sitting duck complex (SDC). SDI at pX occurs when jth EC fired from pY front end reaches the end of the overlapping region, i.e. +1, and encounters an IC stationed at pX , , . SDI is critical or less. We consider the limiting case, where Δcritical = RNAP footprint of an EC (35 bp). Δcritical could be greater than the RNAP footprint when the torsional stress caused by simultaneous uncoiling of both DNA may cause the RNAPs to fall off the DNA [et al. using Atomic Force Microscopy [xk or yh depending on whether the EC originated from pX or pY respectively  when two opposing ECs are separated by critical distance, Δ the DNA . Collisicroscopy . When onectively .k , their degradation rate, λ, and dilution due to growth rate, μ  regulating its strength (fY). Assuming binding of repressor X to operator site OY reaches rapid equilibrium, the fraction of unbound operator sites is given by Equation 17. The RNAP binding rate at pY is considered to be proportional to the concentration of unbound operator sites at pY as described by Equation 18, where fY,min and fY,max are the RNAP firing rates at pY under repressed and de-repressed conditions, respectively. Protein Y implements a positive feedback by indirectly activating production of a transcriptional activator Z, which is considered to bind protein X and relieve repression at pY following a second order kinetics (Equations 15–16). Protein Z may also function as a signaling molecule produced independent of Y activation. We consider Z is also produced from an external source, W, which could be a host gene, a gene from an acquired plasmid, or an environmental signal (released by other cells), following a zero order kinetics, kWZ, (Equation 15). Dissociation of X:Z complex is considered to follow first order kinetics (Equation 16). Parameter nomenclature and their numerical values used are summarized in Based on the architecture of naturally occurring nse gene ,63–66. I systems ,68  and K = 0.8±0.1. For fits of all y switches, kmax = 39.0±7.8 and K = 1.7±0.2. Positive Hill coefficient values indicate increase in RNA production with increase in α whereas, negative values indicate decrease in RNA production with α increase.Response curves were fitted to the form BX = 20 s, τIX = 12 s), whereas pY is an inducible promoter with a variable RNAP binding time (τBY = ατBX), but a fixed RNAP initiation time (τIY = 9.5 s). These parameter values are used hereafter unless otherwise stated. The condition tested here assumes EC’s do not pause and move with an average velocity of 50 bp/s (⟨vx⟩ = ⟨vy⟩ = 50 bp/s) from pX and pY promoters.Biological conditions can cause the relative strengths of pX and pY promoters, α, to change from α<1 to α>1, giving rise to a switch response in gene expression see . For thex and y transcripts in presence of TI alone  to increase by 10.7-fold due to increased RNAP collisions  to play a dominant role that can allow for tuning of full-length transcript expression as well as production of specific truncated RNA distributions. Carefully controlling TI may provide an opportunity to maximize production of specific sizes of truncated transcripts that may have the potential to interact with the antisense RNA ,63,71. Fllisions . As a gellisions  whereas llisions . The oppllisions .gal promoter relative to the PL promoter [k/L>1) are higher from the stronger pX promoter, whereas minimal from the weaker pY promoter (When α is far from 1 (one promoter is much stronger than the neighboring promoter), occlusion is the dominant TI mechanism since high frequency of ECs severely hinders binding of RNAP at the weaker promoter. This is evident in the range of low α values from α = 0.1 to α = 0.4 , where mpromoter . Upon ocpromoter . Althougy transcript are expressed in addition to a narrow truncated RNA size distribution, mainly corresponding to yh transcripts of length L-fp/2 due to SDC survival (xk/L = 0.95) that then decreases by 18.5-fold at α = 2  and 320 bp (xk/L = 0.8). As a result the average velocity of ECs in the sense strand decrease ⟨vx⟩<50 bp/s, whereas ⟨vy⟩ = 50 bp/s. Paused RNAP are treated similar to SDCs initiating at the promoter region, i.e. in a collision event between an EC and a paused RNAP, we consider that the latter will fall off the DNA in 90% of the cases while in the remaining 10% of the cases the ECs will stop transcribing. Interestingly, the broad range of sizes of truncated RNA from pX in absence of RNAP pausing converges to narrower distributions in presence of 2 s long pauses [xk truncated RNA at pause site 160 bp (xk/L = 0.4) and 360 bp (xk/L = 0.8) increase 3.6- and 4.3-fold, respectively. Thus, pausing can be used as design tool to bias production of a truncated RNA predominantly along just one DNA strand. Importantly, such a strategy can be used to design synthetic constructs that express specific truncated RNA products including riboswitches or non-coding RNAs participating in RNA interaction.Previous experimental data indicate that transcriptional pausing is ubiquitous in natural systems and usually occurs at every 100–200 bp for durations of 1–6 s on an average . To evalg pauses  due to ix switch (H = -3.1), however, it caused an increase in the dynamic range of x (between α = 0.1 and 2) from 35-fold for no pause (0 s), to 70-fold for 4 s pause, and 475-fold for 8 s pause. Conversely, for y switch the dynamic range (y at α = 0.1/ y at α = 2) was slightly reduced from 106-fold for no pause (0 s), to 84-fold for both 4 s and 8 s pauses  at the same pause sites on sense strand did not significantly impact the sharpness of the s pauses . Pauses s pauses . In abseollision . The poskx and ky) and truncated RNA species . To eliminate AR, we set the rate constant for RNA interactions in corresponding equations to zero. Again we varied α from α<1 to α>1 by modifying τBY while keeping τIY, τBX and τIX constant. While the expression level of x stayed constant when varying α for the “None” case, x levels changed for AR, TI and TI+AR cases  was observed in the TI+AR case, followed by AR and TI cases, which had similar sharpness . If none of the mechanisms were considered the Hill coefficient was 0 for x as expected because constitutive pX strength is independent of α and 1 for y . It is important to note that hypersensitive switches confer the system with a higher-order response compared to a first-order response characterized by a linear or ramping behavior. As α increases from 0.1 to 2, TI+AR case allows three orders of magnitude change in x levels whereas no change is observed for “None” case. Similarly, y levels for TI+AR case vary three orders of magnitude compared to just one order for “None” case. The switch observed in x and y levels from α = 0.1 to α = 2 is approximately an order of magnitude wider in the TI+AR case when compared to TI only and AR only cases. For the chosen parameters, switch in gene expression is very similar for TI only and AR only cases. It is important to note that the switching response depends on RNA binding, unbinding and degradation rates of the interacting species.We analyzed the four potential combinations of mechanisms that occur during convergent transcription: presence of both TI and AR regulatory mechanisms (TI+AR), exclusively TI (TI), exclusively AR (AR), and none of the mechanisms of TI and AR (None). We eliminated TI by setting the transcription rate of full-length AR cases , despitex and y respectively, exert negative and positive feedback to create a gene regulatory network based on cis-antisense transcription  of transcriptional activator/signaling molecule Z. Steady state solutions show that coupling TI and AR with feedback from regulatory proteins give rise to a characteristic bistable switch response. When bistability is present, cells can adopt two experimentally observable distinct states depending on the production rate of Z  transcriptional parameters, including the ones that affect the TI model and therefore influence the RNAP collision loci and truncated RNA distribution  caused the bistable curve to shift to lower values of kWZ needed to observe the bistable region. Further decrease caused the bistable switch to become irreversible. It is also important to note that relative promoter strength at the de-repressed state of pY  strongly influences the shape of the dose-response curve . As pY at the repressed state gets stronger , bistable range shrinks, at the same time the curve is shifted to lower kWZ values. For fY,min = 1.5X we observe bistability for a kWZ range ~0.6 nM/s  a Michaelian response is observed, where X levels decrease over 2 orders of magnitude as kWZ increases from 0 to 20 nM/s. A 4-fold increase in kbxy (2X) transforms the response into a bistable switch, with X levels corresponding to OFF and ON states separated by 4 orders of magnitude, along with bistable region of nearly 10 kWZ units. Loss of bistability as kbxy decreases suggests that AR is a key regulatory player in order to obtain a bistable switch. This was confirmed by studying the previous None, TI, AR and TI+AR cases in presence of the protein regulation, and observing loss of bistability for None and TI cases . This is exemplified by the pL-pR system of convergent promoters in coliphage 186, where, as a result of SDCs, a 5.6-fold reduction in activity of the weaker lysogenic (pL) promoter was observed, resulting in switching between the lysogenic and lytic states [R-PRE promoter pair of bacteriophage λ where TI from the weaker PRE promoter resulted in a 5.5 fold decrease in expression from the stronger PR promoter due to occlusion [Convergent arrangements of promoters are widespread in archea, bacteria and eukaryotes ,74,75. Afaecalis ,71, undec states . Experimc states ,39,51 alet al. developed stochastic, numerical (mean field) and analytical approaches to fit TI in vivo data on sets of convergent promoters [Previously, Sneppen romoters ; but conet al. [et. al [in trans, which is analogous to the AR only model shown in et al and Mehta et al, thus highlighting the generality of our model. Our model further shows that adding cis-RNA interaction along with TI mechanism can considerably sharpen the switching response with as high as 3 orders of magnitude difference in of full-length RNA levels ; which highlights the opportunity to tweak cellular responses when designing synthetic genetic devices based on cis-antisense transcription. Even for the parameters the system is more sensitive to, majority of them can be modified up to 4-fold without losing bistability. This confers the system with robustness as the noisy fluctuations are not expected to cause changes greater than 10% or 0.1-fold as previously shown in Elowitz et al. [Antisense transcription can result in complex cellular behavior especially in context of a biological gene network. Presence of a feedback loop, implemented in our generalized system by the control of promoter pY strength by protein X encoded from the convergent pX promoter enables the system to behave in a bistable fashion Figs . Such gez et al. .prgQ/prgX operon of E. faecalis [B. subtilis controlling plasmid copy number has been speculated to be regulated by the three regulatory layers presented here: TI, AR and protein regulation of the transcriptional process, but no bistability has been observed [ubiG/mccBA operon of C. acetobutlyicum, both mechanisms of TI and RNA interaction confer a genetic switch regulating the expression of ubiG operon, which contains genes required for conversion of methionine to cysteine [Bordetella [Reversible bistable switches are especially important for their robustness, allowing cells to make important decisions to adapt to new environments such production of antibiotics , plasmidfaecalis ,39 controbserved . In ubiGcysteine . Similarrdetella . Finallyrdetella .cis-antisense RNA not discovered yet that regulate the protein encoded by the antisense gene. Better understanding of TI and AR will provide important insights into biological functioning of microorganisms as well as higher organisms and development of improved biological systems. Given that promoter strength, overlapping DNA sequence and length is highly tunable, antisense transcription could be exploited to tweak naturally existing networks or create novel networks for synthetic biology applications. Moreover, genetic engineering taking advantage of these layers of regulation could allow us to develop engineered cells acting as biological sensors that perform different functions depending on the environmental conditions. Finally, although widespread presence of antisense transcription exists, further experimental characterization studies need to be done to understand whether antisense transcription is a fortuitous phenomenon in in nature or a well-designed set-up for gene regulation that could be controlled even at the genome scale.Recent discovery of many new small RNAs, often with unknown function, indicate the presence of promoters opposite to protein-coding regions –94. MostS1 Table(DOCX)Click here for additional data file.S1 Fig Expression maps of full-length  and truncated ((C) Switch response in full-length x and y transcript levels becomes sharper for lower percentage of RNAP survival, at 80% the Hill coefficient is -3.1 for x and 2.9 for y, at 40% it is -4.0 for x and 4.4 for y, and at 0% it is -7.8 for x and 8.2 for y.(TIFF)Click here for additional data file.S2 Fig Effect of τIX. Maps of truncated and full-length transcripts originating from pX (A) and pY (B), respectively, at different initiation intervals at pX, τIX. Longer initiation times at pX increase probability of sitting duck interference at pX. Dislodgement of more sitting duck complexes at pX cause decreased RNAP collision and lowers expression of full-length x transcripts. (C) The switch response of x and y RNA at different τIX values. x transition becomes sharper at higher τIX values.  Effect of τIY. As the initiation time at pY increases, analogous effect is seen. Sitting duck interference at pY increases and less RNAP collisions occur. (F) Switch response in x and y levels at different τIY values. y levels are lowered at higher τIY.(TIFF)Click here for additional data file.S3 Fig Expression maps of truncated and full-length transcripts originating from pX (A) and pY (B), respectively, at different values of overlapping length, L. Longer overlapping regions yield to increased RNAP collisions. (C) Enhanced collisions lower the expression of full-length x and y transcripts. In this case, no clear trend was observed for the Hill coefficient.(TIFF)Click here for additional data file.S4 Fig Levels of full-length x (A) and y (B) transcripts, respectively, for None, TI, AR and TI+AR cases of antisense transcription in presence of the gene regulatory network implemented by proteins X, Y and Z. Bistability is only observed when AR is present (AR and TI+AR cases). When TI is coupled with AR (TI+AR case) the bistable range is expanded and the switch response is widened.(TIFF)Click here for additional data file.S5 Figλx, degradation rate of full-length x. λy, degradation rate of full-length y.Protein X and Y levels are shown for each modified parameter. Arrows indicate the way the curve shifts as the tuned parameter increases. (TIFF)Click here for additional data file.


There is a critical need for therapeutic agents that can target the amino-terminal domain (NTD) of androgen receptor (AR) for the treatment of castration-resistant prostate cancer (CRPC). Calmodulin (CaM) binds to the AR NTD and regulates AR activity. We discovered that Hydrazinobenzoylcurcumin (HBC), which binds exclusively to CaM, inhibited AR activity. HBC abrogated AR interaction with CaM, suppressed phosphorylation of AR Serine81, and blocked the binding of AR to androgen-response elements. RNA-Seq analysis identified 57 androgen-regulated genes whose expression was significantly (p ≤ 0.002) altered in HBC treated cells as compared to controls. Oncomine analysis revealed that genes repressed by HBC are those that are usually overexpressed in prostate cancer (PCa) and genes stimulated by HBC are those that are often down-regulated in PCa, suggesting a reversing effect of HBC on androgen-regulated gene expression associated with PCa. Ingenuity Pathway Analysis revealed a role of HBC affected genes in cellular functions associated with proliferation and survival. HBC was readily absorbed into the systemic circulation and inhibited the growth of xenografted CRPC tumors in nude mice. These observations demonstrate that HBC inhibits AR activity by targeting the AR NTD and suggest potential usefulness of HBC for effective treatment of CRPC. Prostate cancer (PCa) is the most frequently diagnosed non-skin cancer and second leading cause of cancer related deaths in American men . AndrogeAR belongs to the steroid receptor family of transcription factors containing four distinct functional domains; amino-terminal domain (NTD) containing activation function (AF-1), DNA binding domain (DBD), hinge region (HR) containing nuclear localization sequence, and carboxy-terminal LBD. The AR NTD is crucial for AR transcriptional activity; while AR devoid of LBD can be constitutively active, there can be no transcriptional activity without NTD . A numbeCaM is a ubiquitous calcium-binding protein that has no intrinsic enzymatic activity but binds to and regulates the activity of a variety of enzymes involved in cellular processes such as cell proliferation and viability, and cancer . CaM is Since CaM regulates AR activity , 12, andWe have shown previously that CaM levels are 2- to 3- fold higher in AR-positive LNCaP cells than in AR-negative PC-3 cells . We now 3H-thymidine into DNA, indicating an anti-proliferative effect of HBC on LNCaP cells with an IC50 of ~5 μM  containing medium to hormone-depleted CSS containing medium, resulted in a dramatic decrease in protein levels of AR and PSA Fig. . Treatmein-vitro EMSA showed an inhibitory effect of HBC on the binding of AR to PSA-ARE  in 1,766 transcripts representing 1,475 genes and their isoforms in androgen-deprived cells treated with R1881 in the absence vs. presence of HBC (data not shown). Genes that were altered most significantly (p <0.002) among these were 29 androgen-stimulated genes that were repressed by HBC and 28 androgen-repressed genes that were induced by HBC Table  and, conThe gene expression changes observed in R1881 vs. R1881 + HBC treated cells were validated by RT-qPCR analysis of representative genes Fig. . Among t−21 to 8.7×10−9) functional categories of genes modulated by HBC  to identify HBC affected genes that are likely to be regulated by AR and CaM. IPA identified 84 AR-regulated and 19 CaM-regulated genes among the genes that were affected (p<0.05) by HBC Fig. . In addimax of 16.44 μg/mL at 1 h. HBC exhibited an apparent systemic clearance  similar to the hepatic blood flow in mice (5.4 L/h). HBC was rapidly distributed into tumor tissues  and achieved a tumor drug concentration  similar to the systemic (plasma) drug concentration   Fig. .Based on the pharmacokinetic data, we treated tumor bearing mice with 100 mg/Kg HBC twice daily/5 days/week. This treatment regimen had no significant effect on either the body weight of mice  or on grRecently, p300 acetyltransferase activity has been reported to be crucial for AR-targeted gene expression . InteresCurrent strategies for the treatment of metastatic PCa rely on an intact AR LBD to inhibit AR activity. However, in CRPC, AR LBD is sometimes deleted or mutated rendering AR refractory to all forms of androgen-deprivation and/or AR-antagonists currently available , 29. The50 of 140 μM  in a final volume of 20 μl for 30 min on ice. Samples were then subjected to 6% native polyacrylamide gel electrophoreses, transferred onto positively charged nylon membrane (Roche Applied Science), and PSA-ARE probe signal on nylon membrane was then detected using an anti-DIG-alkaline phosphatase conjugate and chemiluminescence.Total RNA prepared as described above was submitted to Applied Genomics Technology Center, Wayne State University School of Medicine, where RNA integrity was confirmed using Agilent 2000 TapeStation system, sequencing adaptor ligated cDNA libraries were constructed using TruSeq RNA Sample Preparation Kit v2 , and 50 cycle paired-end sequencing was done on Illumina HiSeq 2500. Details of RNA-Seq data analysis can be found in A set of 57 genes Table , whose eFunctional interactions between the set of genes whose expression was either decreased or increased significantly  in cells treated with R1881 + HBC, as compared to R1881 alone, was evaluated using the Ingenuity curated knowledge base as a reference and Ingenuity Pathway Analysis (IPA) tool .6) and measurement of the volume of tumors derived from C4-2B cells in six week old male athymic nude mice was as described previously [3, the tumor-bearing mice were randomly assigned to a solvent-treated (control) or HBC-treated group and given either solvent (100 μl PBS) or HBC intra-peritoneally (i.p.) 100 mg/kg twice daily for 5 consecutive days/week. The experiment was terminated when the control tumors reached ~1,100 mm3 (on day 28) and the mice were sacrificed. Part of each tumor was used to prepare the tissue extracts and the remainder was fixed and paraffin embedded. Tissue extracts for Western blotting were prepared as described previously [The animal protocols were reviewed and approved by the Institutional Laboratory Animal Care and Use Committee of Henry Ford Hospital. Subcutaneous (s.c.) injection of C4-2B cells  were collected at 5 min, and 0.5, 1, 2, 4, 6, and 24 hours, with two mice for each time point. A detailed description of procedures for measuring HBC levels in plasma and tissue samples and pharmacokinetic data analysis can be found in Benign and tumor human prostate tissue sections were obtained with approval from the Institutional Review Board of Henry Ford Health System. Tissue sections were subjected to heat-induced epitope retrieval using DAKO PT LINK Chamber and immunostained on the DAKO LINK Autostainer using EnVision FLEX reagents  by following the manufacturer's suggested protocol. Primary antibodies used for staining were rabbit monoclonal antibodies against CaM (Abcam) diluted 1:3,500, and mouse monoclonal antibodies against AR (DAKO) diluted 1:75. Immunostaining of mouse tissue sections with antibodies against Ki67 and H3 (acetyl K9) was performed manually using reagents from Vector Laboratories  by following the manufacturer's suggested protocol.


Results from decades of research already evidenced a positive relationship between abnormal nerve density and ventricular arrhythmias after MI. In this review, we summarized the molecular mechanisms involved in cardiac sympathetic rejuvenation and mechanisms related to sympathetic hyperinnervation and arrhythmogenesis after MI and analyzed the potential therapeutic implications of nerve sprouting modification for ventricular arrhythmias and SCD control.Ventricular arrhythmogenesis is thought to be a common cause of sudden cardiac death following myocardial infarction (MI). Nerve remodeling as a result of MI is known to be an important genesis of life-threatening arrhythmias. It is hypothesized that neural modulation might serve as a therapeutic option of malignant arrhythmias. In fact, left stellectomy or  The majority of sudden cardiac deaths (SCD) are caused by ventricular tachyarrhythmias (VT) secondary to acute myocardial infarction (MI) , which iThe heart is extensively innervated by the autonomic nervous system which is mainly composed of sympathetic and parasympathetic components . Sympath123I-meta-iodobenzylguanidine (MIBG) studies presented evidence that sympathetic reinnervation exists in the infracted hearts of patients [It has been well known that MI could result in degeneration and death of sympathetic fibers within the scar . In addipatients . There apatients , whereaspatients , 29, 30.patients . Additiopatients , 32, a cpatients , 34. IndIn fact, the sympathetic efferent regeneration effort is triggered by neurotrophic factors, particularly nerve growth factor (NGF). NGF is a potent neural chemoattractant that exerts critical functions for the survival and differentiation of sympathetic neurons and promotes peripheral nervous axon outgrowth , 36. FolAlthough axon pruning of heart during development is critical for establishment of appropriate neural circuitry which is regulated by a number of factors such as ephrins and semaphorins –54, the Overall, as MI results in marked upregulation of neural chemoattractants expression without corresponding elevated chemorepellents expression, it might be that this unbalance in the infracted heart leads to the excessive regeneration behavior of sympathetic nerve axons, thereby contributing to the enhanced risk of ventricular arrhythmias and SCD after MI.+ currents such as Ito, IK1, IKs are downregulated in border-zone cells [IKs blocker, sympathetic stimulation produces an abbreviation of the epicardial and endocardial cells APD but not that of the M cells, resulting in an accentuated dispersion of repolarization and widening of the T wave [IKs is also abnormal, β-adrenergic stimulation with epinephrine could result in torsade de pointes (TdP) [2+ current and increased repolarization dispersion [+ currents [MI could cause important changes in cellular electrical activity  particular of border-zone cells functionally based on ion-channel abnormalities due primarily to ionic loss, membrane breakdown, and intracellular acidosis . For exane cells –63. Genene cells . In the e T wave . In paties (TdP) . Thus maspersion . In a pocurrents . Ajijolacurrents . These dIt is widely accepted that sympathetic remodeling resulting from MI is strongly associated with the development of VT, VF, and SCD , 70, 71.ICa may lengthen APD in myocardium. When IKs was downregulated and induced by MI, sympathetic stimulation tended to accentuate dispersion of repolarization [IK1, Ito and ionotropic glutamate receptors (iGluRs) [The mechanisms, however, underlying this relationship remain incompletely understood. The excessive sympathetic nerves sprouting was closely correlated to increases of local ventricular transmural dispersion of repolarization  and prolrization . Further(iGluRs) , 81, 82,In addition, in ambulatory dogs with NGF to the LSG, AVB, and MI, most of the malignant ventricular arrhythmias were preceded by increased stellate ganglion nerve activity including low-amplitude burst discharge activity (LABDA) and high-amplitude spike discharge activity (HASDA), along with increased nerve sprouting . Han et β-adrenergic receptor (β-AR) by the release of norepinephrine (NE). In fact, Zhou and coworkers found that both deleterious nerve sprouting and significant increase in density of β3-AR occur in dogs with MI, AVB, and NGF infusion to the LSG [β3-AR, presents in canine and human cardiac myocytes and exerts profound functions to mediate the membrane ion currents [β3-AR could decrease slow delayed rectifier K+ current and IKs, contributing to slight prolongation of the APD [β2-AR response. Interestingly, similar responses did not occur before MI [β2-AR activation can provoke increase in intracellular Ca2+ transients and after contractions that ultimately trigger VF in the post-MI animal model [β-AR remodeling in postinfarcted heart is important for formation of a substrate that triggers malignant ventricular arrhythmias and leads to SCD in chronic MI.It is well established that the sympathetic innervation of the heart functions mainly through the activation of  the LSG . β3-AR, currents –88. For  the APD . Additioefore MI . Presumaal model , 90. AltCollectively, excessive sympathetic nerve sprouting to reinnervation myocardium in response to MI may be an important element in the arrhythmogenicity of sympathetic nerves remodeling. On the other hand, modest nerve sprouting may contribute to improved hemodynamic performance of the surviving myocardium  and pote