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Adversarial robustness assessment for video recognition models has raised concerns owing to their wide applications on safety-critical tasks. Compared with images, videos have much high dimension, which brings huge computational costs when generating adversarial videos. This is especially serious for the query-based black-box attacks where gradient estimation for the threat models is usually utilized, and high dimensions will lead to a large number of queries. To mitigate this issue, we propose to simultaneously eliminate the temporal and spatial redundancy within the video to achieve an effective and efficient gradient estimation on the reduced searching space, and thus query number could decrease. To implement this idea, we design the novel Adversarial spatial-temporal Focus (AstFocus) attack on videos, which performs attacks on the simultaneously focused key frames and key regions from the inter-frames and intra-frames in the video. AstFocus attack is based on the cooperative Multi-Agent Reinforcement Learning (MARL) framework. One agent is responsible for selecting key frames, and another agent is responsible for selecting key regions. These two agents are jointly trained by the common rewards received from the black-box threat models to perform a cooperative prediction. By continuously querying, the reduced searching space composed of key frames and key regions is becoming precise, and the whole query number becomes less than that on the original video. Extensive experiments on four mainstream video recognition models and three widely used action recognition datasets demonstrate that the proposed AstFocus attack outperforms the SOTA methods, which is prevenient in fooling rate, query number, time, and perturbation magnitude at the same., Comment: accepted by TPAMI2023

Lung cancer (LC) is the most common malignancy in the world. It is well that hypoxia is common in lung cancer, which contributes to lung cancer progression and metastasis [1]. miRNA-27a as a repressor factor is a lowly expression within non-small cell lung cancer (NSCLC). However, the molecular mechanism between miR-27a and hypoxia in lung cancer progression remains poorly understood. This study aims to explore hypoxia promotes epithelial-mesenchymal transition in lung cancer cells via regulating the NRF2/miR‑27a/BUB1 pathway.We detect the expression of miR-27a after exposure to hypoxia conditions in lung cancer cells via qPCR. Using MTT assay and colony assay to assess the ability of proliferation in lung cancer cells under hypoxia or transfect miR-27a mimics. The capability of migration and invasion was evaluated by wound healing assay and Boyden-chamber assay. The mRNA and protein expression of EMT markers was respectively detected by qPCR and western blot. We detected NRF2 occupancy at the miR-27a promoter by ChIP-Seq analysis. Meanwhile, the luciferase assay verified BUB1 as a direct target of miR-27a.We found hypoxia promotes lung cancer cell proliferation, migration, invasion, and the epithelial-mesenchymal transition (EMT) process by inhibiting the miR-27a expression. miR-27a mimics significantly reduced the promotion effect of hypoxia on the invasion and proliferation of lung cancer cells. NRF2 as regulating the oxidation/anti-oxidation factor was activated under hypoxia conditions. The activation of NRF2 repressed miR-27a expression. On the contrary, the inhibitory effect of hypoxia on miR-27a was reversed when the NFE2L2 gene was silenced. Ectopic expression of NRF2 inhibited miR-27a expression under normoxia. We further validated BUB1 as a direct target of the miR-27a by luciferase assay.Hypoxia promotes invasion and epithelial-mesenchymal transition of Lung cancer cells by regulating the NRF2/miR-27a/BUB1 axis.

Supplementary Figure S1 and Supplementary Table S1.

Purpose: Aberrant DNA methylation changes are common somatic alterations in prostate carcinogenesis. We examined the methylation status of six genes in prostate tissue specimens from African American (AA) and Caucasian (Cau) males.Experimental Design: We used pyrosequencing to quantitatively measure the methylation status of GSTP1, AR, RARβ2, SPARC, TIMP3, and NKX2-5. Real-time PCR was used to determine gene expression, and gene reactivation was analyzed by 5-aza-2′-deoxycytidine and/or trichostatin A treatment.Results: Statistical analysis showed significantly higher methylation in the prostate cancer tissue samples in comparison with matched normal samples for GSTP1 (P = 0.0001 for AA; P = 0.0008 for Cau), RARβ2 (P < 0.001 for AA and Cau), SPARC (P < 0.0001 for AA and Cau), TIMP3 (P < 0.0001 for AA and Cau), and NKX2-5 (P < 0.0001 for AA; P = 0.003 for Cau). Overall, we observed significant differences (P < 0.05) in the methylation level for all genes, except GSTP1, in the AA samples in comparison with the Cau samples. Furthermore, regression analysis revealed significantly higher methylation for NKX2-5 (P = 0.008) and TIMP3 (P = 0.039) in normal prostate tissue samples from AA in comparison with Cau, and a statistically significant association of methylation with age for NKX2-5 (P = 0.03) after adjusting for race.Conclusion: Our findings show higher methylation of several genes in prostate tissue samples from AA in comparison with Cau and may potentially contribute to the racial differences that are observed in prostate cancer pathogenesis. Clin Cancer Res; 16(14); 3539–47. ©2010 AACR.

Purpose: Endoplasmic reticulum (ER) stress regulates mucus hypersecretion, and may activate downstream factors via TBK1 signaling to induce gene expression. However, it remains unclear whether ER stress promotes airway mucus secretion through the TBK1 pathway. We aimed to investigate the role of the TBK1 pathway in the regulation of MUC5AC expression in a mouse model of house dust mite (HDM)-induced allergic asthma. Materials and Methods: Mice with HDM-induced asthma and human bronchial epithelial BEAS-2B cells were treated with amlexanox, an anti-allergy drug (25 μM), or 4-PBA (10 mM). Tissue and cell samples were collected. Tissue samples were stained with hematoxylin and eosin (H&E) or periodic acid Schiff (PAS) to evaluate pathology. Protein expression was analyzed by western blotting and immunofluorescence. Results: Mice exposed to HDM presented ER stress and hypersecretion of mucus Muc5ac from airway epithelial cells (p < 0.001). Similar results were observed in BEAS-2B cells following exposure to HDM. Both in vivo and in vitro studies revealed that HDM-induced ER stress induced MUC5AC overexpression via TBK1 signaling. Amlexanox and 4-PBA markedly reduced mucus production and weakened the TBK1 signal, which mediates MUC5AC hypersecretion. Conclusion: TBK1 plays a pivotal role in HDM-induced ER stress, leading to overproduction of MUC5AC in the asthmatic airway epithelium. The overproduction of MUC5AC can be significantly decreased by inhibiting TBK1 or ER stress using 4-PBA. These findings highlight potential target-specific therapies for patients with chronic allergic asthma.

Background The current study aimed to comprehensively analyze the clinical prognostic factors of malignant esophageal fistula (MEF). Furthermore, this study sought to establish and validate prognostic nomograms incorporating radiomics and clinical factors to predict overall survival and median survival after fistula for patients with MEF. Methods The records of 76 patients with MEF were retrospectively analyzed. A stepwise Cox proportional hazards regression model was employed to screen independent prognostic factors and develop clinical nomograms. Radiomic features were extracted from prefistula CT images and post fistula CT images. Least absolute shrinkage and selection operator (LASSO) regression and Cox regression algorithm was used to filter radiomic features and avoid overfitting. Radiomic signature was a linear combination of optimal features and corresponding coefficients. The joint prognostic nomograms was constructed by radiomic signatures and clinical features. All models were validated by Harrell's concordance index (C‐index), caliberation and bootstrap validation. Results For overall survival, age, prealbumin, KPS and interval between diagnosis of esophageal cancer and fistula were identified as independent prognostic factors and incorporated into the clinical nomogram. Age, prealbumin, serum albumin, KPS and neutrophil proportion were selected for the clinical nomogram of post fistula survival. The C‐index of overall survival nomogram was 0.719 (95% CI: 0.645–0.793) and that was 0.722 (95% CI: 0.653–0.791) in the post fistula survival nomogram. The radiomic signature developed by radiomic features of prefistula CT showed a significant correlation with both overall survival and post fistula survival. The C‐index of joint nomogarm for overall survival and post fistula survival was 0.831 (95% CI: 0.757–0.905) and 0.77 (95% CI: 0.686–0.854), respectively. The calibration curve showed the joint nomograms outperformed the clinical ones. Conclusions The study presents nomograms incorporating independent clinical risk factors and radiomic signature to predict the prognosis of MEF. This prognostic classification system has the potential to guide therapeutic decisions for patients with malignant esophageal fistulas., The study presents nomograms incorporating independent clinical risk factors and radiomic signature to predict the prognosis of malignant esophageal fistula. This risk classification system has the potential to guide therapeutic decisions for patients with malignant esophageal fistula.

Patients with sickle cell disease (SCD) have a lower risk for HIV-1 infection. We reported restriction of ex vivo HIV-1 infection in SCD peripheral blood mononuclear cells (PBMCs) that was due, in part, to the upregulation of antiviral, inflammatory, and hemolytic factors, including heme oxygenase-1 (HO-1). Here, we investigated whether individuals with sickle cell trait (SCT), who develop mild hemolysis, also restrict HIV-1 infection. Ex vivo infection of SCT PBMCs exhibited an approximately twofold reduction of HIV-1 replication and lower levels of HIV-1 reverse transcription products, 2-long terminal repeat circle, HIV-1 integration, and gag RNA expression. SCT PBMCs had higher HO-1 messenger RNA (mRNA) and protein levels and reduced ribonucleotide reductase 2 (RNR2) protein levels. HO-1 inhibition by tin porphyrin eliminated ex vivo HIV-1 restriction. Among Howard University clinic recruits, higher levels of HO-1 and RNR2 mRNA and lower HIV-1 env mRNA levels were found in SCT individuals living with HIV-1. To determine the population-level effect of SCT on HIV-1 prevalence, we assessed SCT among women living with HIV (WLH) in the WIHS (Women Interagency HIV-1 Study). Among WIHS African-American participants, the prevalence of SCT was lower among women with HIV compared with uninfected women (8.7% vs 14.2%; odds ratio, 0.57; 95% confidence interval, 0.36-0.92; P = .020). WIHS WLH with SCT had higher levels of CD4+/CD8+ ratios over 20 years of follow-up (P = .003) than matched WLH without SCT. Together, our findings suggest that HIV-1 restriction factors, including HO-1 and RNR2, might restrict HIV-1 infection among individuals with SCT and limit the pathogenicity of HIV.

Background Distant metastasis, which occurs at a rate of 25% in patients with esophageal cancer (EC), has a poor prognosis, with previous studies reporting an overall survival of only 3–10 months. However, few studies have been conducted to predict distant metastasis in EC, owing to a dearth of reliable biomarkers. The purpose of this study was to develop and validate an accurate model for predicting distant metastasis in patients with EC. Methods A total of 299 EC patients were enrolled and randomly assigned to a training cohort (n = 207) and a validation cohort (n = 92). Logistic univariate and multivariate regression analyses were used to identify clinical independent predictors and create a clinical nomogram. Radiomic features were extracted from contrast-enhanced computed tomography (CT) images taken prior to treatment, and least absolute shrinkage and selection operator (Lasso) regression was used to screen the associated features, which were then used to develop a radiomic signature. Based on the screened features, four machine learning algorithms were used to build radiomics models. The joint nomogram with radiomic signature and clinically independent risk factors was developed using the logical regression algorithm. All models were validated and compared by discrimination, calibration, reclassification, and clinical benefit. Results Multivariable analyses revealed that age, N stage, and degree of pathological differentiation were independent predictors of distant metastasis, and a clinical nomogram incorporating these factors was established. A radiomic signature was developped by a set of sixteen features chosen from 851 radiomic features. The joint nomogram incorporating clinical factors and radiomic signature performed better [AUC(95% CI): 0.827(0.742–0.912)] than the clinical nomogram [AUC(95% CI): 0.731(0.626–0.836)] and radiomics predictive models [AUC(95% CI): 0.754(0.652–0.855), LR algorithms]. Caliberation and decision curve analyses revealed that the radiomics-clinical nomogram outperformed the other models. In comparison to the clinical nomogram, the joint nomogram's NRI was 0.114 (95% CI: 0.075–0.345), and its IDI was 0.071 (95% CI: 0.030–0.112), P = 0.001. Conclusion We developed and validated the first radiomics-clinical nomogram for distant metastasis in EC which may aid clinicians in identifying patients at high risk of distant metastasis.

Due to the directional nature of sp3-hybridized orbitals and the absence of π-orbitals, the oxidative cleavage of the kinetically and thermodynamically stable C(sp3)–C(sp3) bond is extremely difficult and remains scarcely explored. In this work, under the double argument of quantum mechanics (QM) computations and meticulous experiments on our well-designed C–C single bond cleavage mechanism, we discovered a means of photoinduced selective oxidative C(sp3)–C(sp3) bond cleavage in arylamines, easily achieved by simple visible light irradiation using O2 as a benign oxidant under very mild conditions. The utility of our methodology was demonstrated by the C(sp3)–C(sp3) bond cleavage in morpholine/piperazine arylamines with excellent functional group tolerance. Importantly, our methodology is noteworthy, not only in that it does not require any catalysts, but also in that it provides valuable possibilities for the scalable functionalization of clinical drugs and natural products.

Combination antiretroviral therapy (cART) suppresses human immunodeficiency virus-1 (HIV-1) replication but is unable to permanently eradicate HIV-1. Importantly, cART does not target HIV-1 transcription, which is reactivated in latently infected reservoirs, leading to HIV-1 pathogenesis including non-infectious lung, cardiovascular, kidney, and neurodegenerative diseases. To address the limitations of cART and to prevent HIV-1-related pathogenesis, we developed small molecules to target the noncatalytic RVxF-accommodating site of protein phosphatase-1 (PP1) to prevent HIV-1 transcription activation. The PP1 RVxF-accommodating site is critical for the recruitment of regulatory and substrate proteins to PP1. Here, we confirm that our previously developed 1E7-03 compound binds to the PP1 RVxF-accommodating site. Iterative chemical alterations to 1E7-03 furnished a new analogue, HU-1a, with enhanced HIV-1 inhibitory activity and improved metabolic stability compared to 1E7-03. In a Split NanoBit competition assay, HU-1a primarily bound to the PP1 RVxF-accommodating site. In conclusion, our study identified HU-1a as a promising HIV-1 transcription inhibitor and showed that the PP1 RVxF-accommodating site is a potential drug target for the development of novel HIV-1 transcription inhibitors.

Transcription activation of latent human immunodeficiency virus-1 (HIV-1) occurs due to HIV-1 rebound, the interruption of combination antiretroviral therapy or development of drug resistance. Thus, novel HIV-1 inhibitors, targeting HIV-1 transcription are needed. We previously developed an HIV-1 transcription inhibitor, 1E7-03, that binds to the non-catalytic RVxF accommodating site of protein phosphatase 1 (PP1) and inhibits HIV-1 replication in cultured cells and HIV-1 infected humanized mice by impeding PP1 interaction with HIV-1 Tat protein. However, host proteins and regulatory pathways targeted by 1E7-03 that contribute to its overall HIV-1 inhibitory activity remain to be identified. To address this issue, we performed label-free quantitative proteome and phosphoproteome analyses of non-infected and HIV-1 infected CEM T cells that were untreated or treated with 1E7-03. 1E7-03 significantly reprogramed the phosphorylation profile of proteins including PPARα/RXRα, TGF-β and PKR pathways. Phosphorylation of nucleophosmin (NPM1) at Ser-125 residue in PPARα/RXRα pathway was significantly reduced (20-fold, p=1.37×10

The surfaces of cells and pathogens are covered with short polymers of sugars known as glycans. Complex N-glycans have a core of three mannose sugars with distal repeats of N-acetylglucosamine and galactose sugars terminating with sialic acid (SA). Long-range tough and short-range brittle self-adhesions were observed between SA and mannose residues, respectively, in ill-defined artificial monolayers. We investigated if and how these adhesions translate when the residues are presented in N-glycan architecture with SA at the surface and mannose at the core and with other glycan sugars. Two pseudotyped viruses with complex N-glycan shields were brought together in force spectroscopy (FS). At higher ramp rates, slime-like adhesions were observed between the shields, whereas Velcro-like adhesions were observed at lower rates. The higher approach rates compress the virus as a whole, and the self-adhesion between the surface SA is sampled. At the lower ramp rates, however, the complex glycan shield is penetrated and adhesion from the mannose core is accessed. The slime-like and Velcro-like adhesions were lost when SA and mannose were cleaved, respectively. While virus self-adhesion in forced contact was modulated by glycan penetrability, the self-aggregation of the freely diffusing virus was only determined by the surface sugar. Mannose-terminal viruses self-aggregated in solution, and SA-terminal ones required Ca(2+) ions to self-aggregate. Viruses with galactose or N-acetylglucosamine surfaces did not self-aggregate, irrespective of whether or not a mannose core was present below the N-acetylglucosamine surface. Well-defined rules appear to govern the self-adhesion and -aggregation of N-glycosylated surfaces, regardless of whether the sugars are presented in an ill-defined monolayer, or N-glycan, or even polymer architecture.

It is almost a consensus that decarbonylation of the aldehyde group (–CHO) needs to not only be mediated by transition metal catalysts, but also requires severe reaction conditions (high temperature and long reaction time). In this work, inspired by the “conformational-selectivity-based” design strategy, we broke this consensus and discovered a catalyst-free photodecarbonylation of the aldehyde group. It revealed that decarbonylation can be easily achieved with visible light irradiation by introducing a tertiary amine into the ortho-position of the aldehyde group. A diverse array of tertiary amines is tolerated by our photodecarbonylation under mild conditions. Furthermore, the (QM) computations of the mechanism and the experiments on well-designed special substrates revealed that our photodecarbonylation depends on the conformational specificity of the aldehyde group and tertiary amine, and occurs through an unusual [1,4]-H shift and a subsequent [1,3]-H shift.

Asthma is a chronic heterogeneous inflammatory disease. Most neutrophilic asthma (NA) cases are severe asthma involving many inflammatory cells and mediators, although the specific pathogenesis is not clear. Mucosal-associated invariant T (MAIT) cells as innate-like T lymphocytes play an important role in the immune response in asthma by producing cytokines. In this study, we evaluated the phenotype and function of circulating MAIT cells in patients with NA and inflammatory-related cytokines in plasma and induced sputum supernatants using flow cytometry. The results showed that the frequency of circulating MAIT cells in asthma patients, particularly NA patients, decreased significantly, and CD8

The HIV-1 Tat protein interacts with TAR RNA and recruits CDK9/cyclin T1 and other host factors to induce HIV-1 transcription. Thus, Tat–TAR RNA interaction, which is unique for HIV-1, represents an attractive target for anti-HIV-1 therapeutics. To target Tat–TAR RNA interaction, we used a crystal structure of acetylpromazine bound to the bulge of TAR RNA, to dock compounds from the Enamine database containing over two million individual compounds. The docking procedure identified 173 compounds that were further analyzed for the inhibition of HIV-1 infection. The top ten inhibitory compounds with IC50 ≤ 6 µM were selected and the three least toxic compounds, T6780107 (IC50 = 2.97 μM), T0516-4834 (IC50 = 0.2 μM) and T5628834 (IC50 = 3.46 μM), were further tested for HIV-1 transcription inhibition. Only the T0516-4834 compound showed selective inhibition of Tat-induced HIV-1 transcription, whereas the T6780107 compound inhibited equally basal and Tat-induced transcription and the T5628834 compound only inhibited basal HIV-1 transcription. The compounds were tested for the inhibition of translation and showed minimal (&lt, 25%) effect. The T0516-4834 compound also showed the strongest inhibition of HIV-1 RNA expression and p24 production in CEM T cells and peripheral blood mononuclear cells infected with HIV-1 IIIB. Of the three compounds, only the T0516-4834 compound significantly disrupted Tat–TAR RNA interaction. Additionally, of the three tested compounds, T5628834 and, to a lesser extent, T0516-4834 disrupted Tat–CDK9/cyclin T1 interaction. None of the three compounds showed significant inhibition of the cellular CDK9 and cyclin T1 levels. In silico modelling showed that the T0516-4834 compound interacted with TAR RNA by binding to the bulge formed by U23, U25, C39, G26,C39 and U40 residues. Taken together, our study identified a novel benzoxazole compound that disrupted Tat–TAR RNA interaction and inhibited Tat-induced transcription and HIV-1 infection, suggesting that this compound might serve as a new lead for anti-HIV-1 therapeutics.

Background: The coronavirus disease 2019(COVID-19) had become an epidemic and spread across the world, lead to severe respiratory failure and death. Traditional Chinese Medicine(TCM), Such as Lianhua Qingwen has been widely used in the prevention and treatment of COVID-19, This systematic review and meta-analysis will assess the effects of traditional Chinese herbal medicine(Lianhua Qingwen) in COVID-19 pneumonia from the randomized controlled trials(RCTs) and case control studies(CCSs).Method: we search the literatures in databases inculding PubMed, Embase, Web of science, Cochrane Library, Wanfang, Chinese Science and Technology Periodical Database (VIP), Chinese Biomedical Literature Database (CBM) and China National Knowledge Infrastructure(CNKI), setting the date from December 1, 2019, to June 1, 2021, Cochrane Risk of Bias tool and the Newcastle-Ottawa Scale were used to assess the quality of randomized controlled trials. All analyses were conducted by Stata 14.0.Results: nine studies with 1163 patients(616males) were included, six were RCTs, three are CCSs. Compared with patients treated by western medicine alone, patients treated by Lianhua Qingwen combined with western medicine have a higher overall effective rate[RR=1.20, 95%CI1.20(1.11, 1.31), P=0.000], cardinal symptom disappearance rate[disappearance rate of fever: OR:3.64, 95%CI(1.57, 8.47), P=0.001;disappearance rate of cough: OR:1.97, 95%CI(1.45, 2.68), P=0.001; disappearance rate of fatigue: OR:2.55, 95%CI(1.09, 5.99), P=0.032] and CT recovery rate[RR:1.25, 95%CI(1.13,1.38), P=0.000], reduce the rate of the progress into severe diseases of COVID-19 patients[RR:0.43, 95%CI(0.30, 0.62), P=0.000], with more shorter duration of fever[WMD=-1.07，95%CI(-1.77, -0.37), P=0.003], The including studies described that Lianhua Qingwen did not increase the adverse drug reactions.Conclusion: Lianhua Qingwen may have advantages in improving the clinical effective rate and cardinal symptom disappearance rate. Besides, it also had an excellent effect on the improvement of the chest CT and the proportion reducing of progress into severe clinical disease, which could be used as an effective therapy for COVID-19.

BACKGROUND We hypothesized that ancestry-mediated methylated DNA changes may drive racial and ethnic disparity in prostate cancer (PCa). To test this hypothesis, we analyzed genetic ancestry and association with DNA methylation changes in PCa disparity. MATERIALS AND METHODS Pyrosequencing and ancestry informative markers were used for DNA methylation and genetic ancestry testing, respectively. RESULTS Using Spearman rho rank correlation test, the data demonstrated significant (p

Combination antiretroviral therapy (cART) suppresses human immunodeficiency virus-1 (HIV-1) replication but is unable to permanently eradicate HIV-1. Importantly, cART does not target HIV-1 transcription, which is reactivated in latently infected reservoirs, leading to HIV-1 pathogenesis including non-infectious lung, cardiovascular, kidney, and neurodegenerative diseases. To address the limitations of cART and to prevent HIV-1-related pathogenesis, we developed small molecules to target the noncatalytic RVxF-accommodating site of protein phosphatase-1 (PP1) to prevent HIV-1 transcription activation. The PP1 RVxF-accommodating site is critical for the recruitment of regulatory and substrate proteins to PP1. Here, we confirm that our previously developed 1E7-03 compound binds to the PP1 RVxF-accommodating site. Iterative chemical alterations to 1E7-03 furnished a new analogue, HU-1a, with enhanced HIV-1 inhibitory activity and improved metabolic stability compared to 1E7-03. In a Split NanoBit competition assay, HU-1a primarily bound to the PP1 RVxF-accommodating site. In conclusion, our study identified HU-1a as a promising HIV-1 transcription inhibitor and showed that the PP1 RVxF-accommodating site is a potential drug target for the development of novel HIV-1 transcription inhibitors.

Background Exposed to house dust mite (HDM) is known to be associated with allergic asthma. Endoplasmic reticulum (ER) stress is involved in the regulation of mucus hyper secretion. However, the mechanism remains unclear. Our main goal was to investigate the relationship of TBK1 pathway regulates the expression of MUC5AC under the HDM induced ER stress promote allergic asthma. Methods Using HDM induced allergic mice and stimulated over expression of MUC5AC in human airway epithelial cells. We examined the mucus and the expression of ER stress markers both in vivo and in vitro. Additionally, we investigated whether TBK1, NF-κB, STAT6 play an important role in the HDM induced ER stress promote airway mucus hyper secretion. Results Mice exposed to HDM were identified ER stress, hyper secretion of mucus, and activated TBK1-NF-κB/STAT6 signaling pathway in airway epithelial cells of asthmatic mice. Similarly results had also been observed in the human airway epithelial cells after exposed to HDM. Both in vivo and in vitro study not only revealed that an anti-allergy drug, Amlexanox, reduced super response of mucus and weaken TBK1-NF-κB/STAT6 signal of induced asthma. But also indicated HDM induced ER stress result in over production of MUC5AC, which can be decreased by the inhibition of TBK1, NF-κB, STAT6, or even by using ER stress inhibitor 4-PBA, respectively. Conclusions Our results show that TBK1-NF-κB/STAT6 plays a pivotal role in the HDM induced ER stress result in over production of mucus protein MUC5AC in the asthmatic airway.

The surfaces of cells and pathogens are covered with short polymers of sugars known as glycans. Complex N-glycans have a core of three mannose sugars, with distal repeats of N-acetylglucosamine and galactose sugars terminating with sialic acid (SA). Long-range slime-like and short-range Velcro-like self-adhesions were observed between SA and mannose residues, respectively, in ill-defined monolayers. We investigated if and how these adhesions translate when SA and mannose residues are presented in complex N-glycan shields on two pseudo-typed viruses brought together in force spectroscopy (FS). Slime-like adhesions were observed between the shields at higher ramp rates, whereas Velcro-like adhesions were observed at lower rates. The complex glycan shield appears penetrable at the lower ramp rates allowing the adhesion from the mannose core to be accessed; whereas the whole virus appears compressed at higher rates permitting only surface SA adhesions to be sampled. The slime-like and velcro-like adhesions were lost when SA and mannose, respectively, were cleaved with glycosidases. While virus self-adhesion in FS was modulated by glycan penetrability, virus self-aggregation in solution was only determined by the surface sugar. Mannose-terminal viruses self-aggregated in solution, while SA-terminal ones required Ca2+ions to self-aggregate. Viruses with galactose or N-acetylglucosamine surfaces did not self-aggregate, irrespective of whether or not a mannose core was present below the N-acetylglucosamine surface. Well-defined rules appear to govern the self-adhesion and -aggregation of N-glycosylated surfaces, regardless of whether the sugars are presented in ill-defined monolayer, or N-glycan, or even polymer architecture.

BACKGROUND/AIM: Androgens are essential for the growth of most prostate cancers (PCa). As a result, androgen ablation is the mainstay of the treatment of PCa. Proteins of the polycomb and trithorax family are master epigenetic regulators of cell type specific gene expression including androgen receptor. MATERIALS AND METHODS: We interrogated epigenetic changes of a 24-gene panel corresponding to polycomb and trithorax genes by PCR array and differential gene expression by quantitative real time-PCR on prostate cancer cell line (LNCaP) treated with the synthetic ligand R1881. RESULTS: We observed the highest methylation for CBX2, PCGF6, PHC2, EZH2 and TRIM27 genes and the lowest methylation for CBX8 and PCGF2 (p

LncRNAs are increasingly verified to be aberrantly expressed in cancers. The abnormal expression of lncRNAs has turned out to be closely related to tumorigenesis or tumor progression. It has been reported that lncRNA OPI5-AS1 serves as a crucial regulator in tumors. However, the specific function of OIP5-AS1 on the progression of lung adenocarcinoma is still uncertain. In this paper, we mainly elucidated that OIP5-AS1 exerts oncogenic functions in human lung adenocarcinoma through targeting miR-448. We inspected that the expression of OIP5-AS1 was definitely high in lung adenocarcinoma tissues and cells, while miR-448 was sluggishly expressed in lung adenocarcinoma. OIP5-AS1 and miR-448 was negatively related to each other, the result was obtained from Pearson correlation analysis. We discovered a fact that OIP5-AS1 could directly sponge miR-448 through using dual luciferase reporter assay, RIP assay and RNA pull-down assay. Cell proliferation, migration and invasion were restrained after we disrupted the expression of OIP5-AS1 in lung adenocarcinoma. We also certified that OIP5-AS1 could sponge and regulate miR-448 to affect cell function in lung adenocarcinoma. MiR-448 could target Bcl-2 and affect the expression of Bcl-2. Then, we discovered that the expression of OIP5-AS1 and Bcl-2 was positively related. So we affirmed that lncRNA OIP5-AS1 modulated the expression of Bcl-2 by targeting miR-448 in lung adenocarcinoma cells.

Introduction Patients with Sickle cell disease (SCD) have lower risk for HIV-1 infection. We showed that ex vivo HIV-1 replication is blocked in SCD PBMCs in part because of the increased expression of ferroportin (FPN) and activation of SAMHD1, a host antiviral restriction factor. We hypothesized that rupture of sickling red blood cells releases sickle cell hemoglobin (HbS) that is phagocyted by macrophages leading to upregulation of innate antiviral response and inhibition of HIV-1 replication. We accessed changes in antiviral gene expression in PBMCs obtained from SCD patients compared to healthy controls. We also analyzed antiviral gene expression in macrophages treated with HbS compared to the HbA treatment. Methods The study was approved by Howard University review board (IRB) and all subjects consented to sample collection. Whole blood was collected from 9 SCA patients and 9 age and gender- matched healthy controls. PBMCs were activated with PHA (0.5 μg/ml) for 24-48 hrs followed by IL-2 (10 U/ml) for 24 hrs. Human THP-1 cells were differentiated into macrophages with PMA (25nM) for 72 hrs and treated with purified HbS or HbA (5µM). RNA strand-specific libraries were constructed using TruSeq Stranded Total RNA Gold kit (Illumina) and sequenced on an Illumina NextSeq 500 using 75 bp paired-end sequencing on two v2.5 150 cycle High-Output kits, generating 40-50 million paired-end reads per sample. The sequencing data were mapped using Dragen RNA and compared using Dragen differential expression software (Illumina). Ingenuity Pathway analysis (IPA, Qiagen) was used for pathway analysis. Results In activated SCD PBMCs compared to control PBMCs, 40432 genes were detected including 2230 differentially expressed genes (5.5%, 1.5-fold difference, 287 down and 1943 up) at 5% false discovery rate. In non-activated SCD PBMCs compared to control PBMCs, 33119 genes were detected including 5299 differentially expressed genes (16%, 923 down and 4376 up). In THP-1-differentiated macrophages treated with HbS versus HbA, 28362 genes were detected including 322 differentially expressed genes (1.1%, 187 down and 135 up). We focused our analysis on 61 genes including viral restriction factors and iron regulatory genes. In activated SCD PBMCs, four genes had highest upregulation: APOBEC3A (23-fold, p=2 x 10 -5), CH25H (11-fold, p=4 x 10 -5), heme oxygenase-1 (HMOX1, 13-fold, p=1.5 x 10 -12) and FPN (SLC40A1, 5-fold, p = 9 x 10 -8) (Fig.1). Several additional genes were upregulated with 1.5-3-fold increase and high significance including APOBEC3B, BRD4, CD40, CDKN1A (p21), GDR1, IFIT3, IFITM3 and SAMHD1. In non-activated SCD PBMCs, the most upregulated gene was PKR (EIF2AK2, 15-fold, p=1 x 10 -11) and genes with 1.5-3 fold upregulation included APOBEC3B, BST2, CPSF6, IFI16, IFITM3, ISG15, LGALS3BP, PML and RTF1 (Fig.1). Of these genes, only IFITM3 overlapped between activated and non-activated PBMCs. To test whether circulating HbS leads to the upregulation of antiviral response, we analyzed HbS-treated macrophages and found upregulation of several antiviral restriction factors (1.5-2.3 fold): IFIT3, LGALS3BP, MX2 and RTF1 (Fig.1). Unsupervised IPA showed upregulation of IRF-7 signaling pathway and down regulation of viral infection and replication (Fig.2). We validated the CH25 and HO-1 antiviral role in activated SCD PBMCs using small molecule inhibitors. We also confirmed overexpression of CH25H and HO-1 by western blot and ELISA. We observed higher levels of IRF7 in the activated SCD PBMCs confirming that it may play a role in the induction of antiviral response. Conclusion We propose that HbS released by hemolysis and uptaken by macrophages leads to the IRF-7-triggered induction of antiviral state in macrophages that will induced antiviral state in non-activated circulating PBMCs likely though the cytokines and interferons secretion known to be elevated in SCD patients. Upregulation of PKR (EIFAK2) levels in non-activated PBMCs strongly argue toward this possibility. Upon activation of PBMCs, additional factors are expressed including CH25H, HO-1, APOBEC3A and FPN that facilitated stronger and more robust anti-HIV-1 effect and block viral replication. Taken together, our study point to novel mechanism of upregulation of antiviral factors mediated by sickle cell hemoglobin that included induction of antiviral, heme- and iron- regulatory pathways. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

The synergistic effect of pH and heating on the structure, aggregation behaviour and gel properties of myofibrillar protein (MP) in mirror carp (Cyprinus carpio) was evaluated. The surface hydrophobicity of the control at pH 5.0 (143.6 ± 0.3 μg) was significantly higher than that of other samples (P 0.05). Under the same pH conditions, the decrease in total sulfhydryl content of all samples during the heating process demonstrated that covalent/non-covalent cross-linking occurred between proteins due to heat input. Moreover, the decrease in solubility and the increase in turbidity of all samples verified the fact of MP aggregation, and the changes in the elasticity index (EI) and macroscopic viscosity index (MVI) also indicated a decrease in MP fluidity upon heating treatment. Therefore, the aggregation of MP was affected by pH and heating, and the optimal three-dimensional network structure and gel properties could be formed at pH 6.0 and above 70 °C.

Concerted action of numerous positively acting cellular factors is essential for Human immunodeficiency virus type 1 (HIV-1) replication but in turn is challenged by anti-viral restriction factors. Previously we showed that ex vivo one round HIV-1 replication and replication of fully competent T-tropic HIV-1(IIIB) is significantly reduced in peripheral blood mononuclear cells (PBMCs) obtained from patients with Sickle Cell Disease (SCD). Further, we identified and confirmed CDKN1A (p21) and CH25H as host restriction factors expressed in SCD PBMCs that may contribute to the HIV-1 inhibition, in addition to the previously reported SAMHD1 and IKBα. Since CH25H is an interferon stimulated gene (ISG), we analyzed IRFs and interferon expression in SCD PBMCs. Higher levels of IRF7 and IFNβ mRNA were observed in SCD PBMCs compared to controls. We probed further to ascertain if hemin or sickle Hb was responsible for interferon response. We found upregulation of IFNβ in THP-1 - derived macrophages treated with lysates of HbSS RBCs or purified HbS as compared to untreated or HbA treated controls. HbSS RBCs lysates and purified HbS inhibited HIV-1 gag mRNA expression in monocyte-derived macrophages infected with HIV-1(Ba-L). Recent clinical study showed increased levels of CD4 in HIV-1 infected SCD patients in Africa. Thus we analyzed CD4 levels in HIV-1 IIIB infected SCD PBMCs, and found them to be higher compared to controls. Levels of HIV-1 nef mRNA, that controls CD4 expression was lower in HIV-1 IIIB infected SCD PBMCs. As Nef counteracts SERINC3/5 restriction factor, we analyzed its expression as well as the expression of AP2 clathrin adaptor that is required for Nef mediated internalization of CD4. AP2 expression was lower and SERINC5 expression was higher in SCD PBMCs. CONCLUSIONS: SCD PBMCs could resist HIV-1 infection because of the increased IFNβ production by macrophages exposed to HbSS or sickle cell RBCs. SCD PBMC have increased levels of SERNIC5 and lower levels of HIV-1 Nef and host AP2 expression that, culumlatively, can increased CD4 levels and lead to the overall improved immunological health of SCD patients. ACKNOWLEDGMENTS: This work was supported by NIH Research Grants (1P50HL118006, 1R01HL125005, 1SC1HL150685, 5U54MD007597, 1UM1AI26617 and P30AI087714). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Disclosures No relevant conflicts of interest to declare.

Objective: Esophageal fistula is a critical and fatal complication of esophageal cancer. The aim of this meta-analysis was to explore the risk factors for esophageal perforation in esophageal cancer patients treated with radiotherapy. Methods: Data from the PubMed and Embase databases were retrieved for clinical research published between 1990 and 2018. The Newcastle-Ottawa Scale was used to evaluate the quality of the articles. A meta-analysis was performed using the RevMan 5.3 software provided by the Cochrane Collaboration Network. Results: Seventeen articles were eligible for the meta-analysis. In these articles, over 35 risk factors for esophageal fistula formation were described and 17 risk factors were analyzed. Significant differences in the odds of developing an esophageal perforation were found with regard to age (OR 2.34, 95% CI 1.08–5.03, p = 0.001), ulcerative type (OR 2.72, 95% CI 1.43–5.16, p = 0.002), histology (OR 4.16, 95% CI 1.14–15.12, p = 0.03), T stage (OR 2.66, 95% CI 1.44–4.91, p = 0.002), short-term response (OR 2.21, 95% CI 1.06–4.62, p = 0.03), chemotherapy regimen (OR 2.80, 95% CI 1.38–5.68, p = 0.005), and stenosis (OR 2.00, 95% CI 1.03–3.89, p = 0.04). Conclusions: An age of

e16071 Background: Distant metastasis with an incidence of 25% in esophageal cancer(EC) represents a poor prognosis. However, there was few study for prediction of distant metastasis in EC, due to unsatisfactory specificity of clinical factors and lack of reliable biomarkers. Methods: Two hundred and ninety-nine patients were enrolled and randomly assigned to a training cohort(n = 207) and a validation cohort(n = 92). Logistic univariate and multivariate regression analyses were used to identify clinical independent predictive factors and construct a clinical nomogram. Radiomic features were extracted from contrast-enhanced CT performed before treatment, and Lasso regression was used to screen the optimal features, which were developed a radiomics signature subsequently. Four machine learning algorithms were used to establish radiomics models respectively based on the screened features. The joint nomogram incorporating radiomics signature and clinical independent predictors was developed by logical regression algorithm. All models were further validated by discrimination,caliberation, reclassification and clinical usefulness. Results: The joint nomogram had a better performance [AUC(95%CI): 0.827(0.742-0.912)] than clinical nomogram [AUC(95%CI): 0.731(0.626-0.836)]and radiomics predictive models[AUC(95%CI): 0.747(0.642-0.851),SVM algorithms]. Caliberation curve, and decision curve analysis also revealed joint nomogram outperformed the other models. Compared with the clinical nomogram, net reclassification Improvement(NRI) of the joint nomogram was improved by 0.114(0.075, 0.345),and integrated discrimination Improvement (IDI) was improved by 0.071(0.030-0.112), P= 0.001. Conclusions: We constructed and validated the first joint nomogram for distant metastasis in EC based on radiomics signature and clinical independent predictive factors, which could help clinicians to identify patients with high risk of distant metastasis.

Sex steroid hormones have been reported to induce inflammation causing dysregulation of cytokines in prostate cancer cells. However, the underlying epigenetic mechanism has not well been studied. The objective of this study was to evaluate the effect of sex steroid hormones on epigenetic DNA methylation changes in prostate cancer cells using a signature PCR methylation array panel that correspond to 96 genes with biological function in the human inflammatory and autoimmune signals in prostate cancer. Of the 96-gene panel, 32 genes showed at least 10% differentially methylation level in response to hormonal treatment when compared to untreated cells. Genes that were hypomethylated included CXCL12, CXCL5, CCL25, IL1F8, IL13RAI, STAT5A, CXCR4 and TLR5; and genes that were hypermethylated included ELA2, TOLLIP, LAG3, CD276 and MALT1. Quantitative RT-PCR analysis of select genes represented in a cytokine expression array panel showed inverse association between DNA methylation and gene expression for TOLLIP, CXCL5, CCL18 and IL5 genes and treatment of prostate cancer cells with 5'-aza-2'-deoxycytidine with or without trichostatin A induced up-regulation of TOLLIP expression. Further analysis of relative gene expression of matched prostate cancer tissues when compared to benign tissues from individual patients with prostate cancer showed increased and significant expression for CCL18 (2.6-fold; p0.001), a modest yet significant increase in IL5 expression (1.17-fold; p=0.015), and a modest increase in CXCL5 expression (1.4-fold; p=0.25). In conclusion, our studies demonstrate that sex steroid hormones can induce aberrant gene expression via differential methylation changes in prostate carcinogenesis.

A rapid and sensitive liquid chromatography tandem mass spectrometry (LC/MS/MS) method was developed and validated using spinasterol as the internal standard (IS) for the simultaneous determination of shionone and epi-friedelinol in rat plasma. Plasma samples were pretreated using liquid-liquid extraction with ethyl ether. Chromatographic separation was achieved on a C18 column (100 × 2.1 mm, 5 μm) with an isocratic elution consisting of acetonitrile-0.1% formic acid water (75:25, v/v) at a flow rate of 0.30 mL/min. Detection was performed under the selected reaction monitoring scan using an electrospray ionization in the positive ion mode. The mass transitions were as follows: m/z 427.4 → 95.1 for shionone, m/z 411.4 → 205.2 for epi-friedelinol and m/z 395.3 → 105.2 for IS. All calibration curves exhibited good linearity (r > 0.995) over the concentration range for both components. The intra- and inter-day precisions at three QC and lower limit of quantitation levels were both

Introduction: Transfusional iron (Fe) overload is a significant problem among patients with chronic, transfusion-dependent anemias. Iron overload is an important problem in pediatric sickle cell disease (SCD) patients on chronic transfusion regimens predominantly for primary and secondary prevention of stroke. Coexistent hereditary iron overload conditions contribute to the iron overload phenotype in SCD. For example, the Q248H mutation (rs11568350) in SLC40A1, which encodes ferroportin (FPN), is associated with a mild tendency to increase serum ferritin in the general population and with increased ferritin levels in SCD patients. Nevertheless, the molecular mechanisms underlying the progression to iron overload in SCD patients are poorly understood and more sensitive markers for outcome prediction that can be applied at early clinical stages are lacking. We hypothesize that genetic variation modifies the risk for iron overload in SCD patients and seek to validate previously identified mutation and identify novel genetic markers of iron overload among participants from TOPMed SCD cohorts by performing whole genome sequencing (WGS) association analyses. Methods: The WGS was performed by several national sequencing centers sponsored by NHLBI's TOPMed program at an average depth of 30× using DNA from SCD patient blood samples. Variant calling was performed jointly across TOPMed studies for all samples using the GotCloud pipeline by the TOPMed Informatics Research Center. The TOPMed data Coordinating Center performed quality control for sample identity. The data across the following studies were shared through the database of Genotype and Phenotype (dbGaP) exchange area: Howard PUSH SCD (N=370), OMG SCD (N=636), Walk PHaSST SCD (N=381) and REDS-III Brazil SCD (N=2620) with a total sample size of 4007. The study was approved by the appropriate institutional review boards (IRB) and informed consent was obtained from all participants. Genome Wide Association Analysis of iron overload was carried out using the University of Michigan ENCORE server. We performed single variant tests to test the association of log-transformed serum ferritin levels with single nucleotide variants (SNVs) while adjusting for sex, age, self-reported race, numbers of lifetime red blood transfusions and genetic substructure (PC's 1-10). We used a significance threshold of p Results: We first included PUSH SCD, OMG SCD and Walk PHaSST SCD cohorts with 840 serum ferritin samples in the WGS association analyses, which revealed at the genome-wide level a new rare variant rs137929759 (chr7:49538810 (GRCh38.p12), MAF=0.0043532, p=2.25×10−8). A few variants such as rs80097634 in gene AL163195.3 and RNASE11 (Chr14:20579417. MAF=0.050373, p=3.1×10-7) were close to the genome-wide significance level. We confirmed previously identified associations in SLC40A1 for ferritin (rs11568350, Chr2:189565370, MAF = 0.16853, p = 5.2×10−4). We also found several variants in AC105411.1, TJP1 and DCC that were close to genome-wide significance level. Further analysis will be carried out on the cloud-based platform provided by NHLBI BioData Catalyst using data from all the four cohorts to validate the previous analysis and expand to related phenotype such as transferrin, iron-overload status. Discussion: In this study we identified common and rare variants that associate with serum ferritin concentration. The results from this pilot study point to novel gene variants that might contribute to iron overload in SCD patients and serve as new biomarkers. Future analysis is needed to determine whether the identified variants can also help with therapeutics and outcome prediction for early stages of SCD-associated iron overload. Our findings will be useful for the future treatment of SCD patients and design of novel SCD therapeutics. ACKNOWLEDGMENTS: This work was supported by NIH Research Grants (1P50HL118006, and 1R01HL125005). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Disclosures Gordeuk: Imara: Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; CSL Behring: Consultancy, Research Funding; Ironwood: Research Funding; Novartis: Consultancy. Telen:CSL Behring: Membership on an entity's Board of Directors or advisory committees, Research Funding; Forma Therapeutics: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; GlycoMimetics Inc.: Consultancy.

Long non-coding RNA (lncRNA) is actively transcribed from human genome and has been considered to participate in many processes of various cancers. The purpose of this study was to investigate the expression of LncRNA AFAP1-AS1 and its prognostic value in NSCLC. LncRNA AFAP1-AS1 expression was detected by qRT-PCR which demonstrated that the expression was significantly increased in tumor tissues compared with the adjacent non-cancerous tissues and healthy tissues. The clinical stage, smoking history, infiltration degree, lymph node metastasis and distant metastasis were all proved to impact the expression of LncRNA AFAP1-AS1 (P

Increasing evidence suggests that aberrant DNA methylation changes may contribute to prostate cancer (PCa) ethnic disparity. To comprehensively identify DNA methylation alterations in PCa disparity, we used the Illumina 450K methylation platform to interrogate the methylation status of 485,577 CpG sites focusing on gene-associated regions of the human genome. Genomic DNA from African-American (AA; 7 normal and 3 cancers) and Caucasian (Cau; 8 normal and 3 cancers) was used in the analysis. Hierarchical clustering analysis identified probe-sets unique to AA and Cau samples, as well as common to both. We selected 25 promoter-associated novel CpG sites most differentially methylated by race (fold change > 1.5-fold; adjusted P < 0.05) and compared the β-value of these sites provided by the Illumina, Inc. array with quantitative methylation obtained by pyrosequencing in 7 prostate cell lines. We found very good concordance of the methylation levels between β-value and pyrosequencing. Gene expression analysis using qRT-PCR in a subset of 8 genes after treatment with 5-aza-2′-deoxycytidine and/or trichostatin showed up-regulation of gene expression in PCa cells. Quantitative analysis of 4 genes, SNRPN, SHANK2, MST1R, and ABCG5, in matched normal and PCa tissues derived from AA and Cau PCa patients demonstrated differential promoter methylation and concomitant differences in mRNA expression in prostate tissues from AA vs. Cau. Regression analysis in normal and PCa tissues as a function of race showed significantly higher methylation prevalence for SNRPN (P = 0.012), MST1R (P = 0.038), and ABCG5 (P < 0.0002) for AA vs. Cau samples. We selected the ABCG5 and SNRPN genes and verified their biological functions by Western blot analysis and siRNA gene knockout effects on cell proliferation and invasion in 4 PCa cell lines (2 AA and 2 Cau patients-derived lines). Knockdown of either ABCG5 or SNRPN resulted in a significant decrease in both invasion and proliferation in Cau PCa cell lines but we did not observe these remarkable loss-of-function effects in AA PCa cell lines. Our study demonstrates how differential genome-wide DNA methylation levels influence gene expression and biological functions in AA and Cau PCa.

Prostate cancer (PCa) harbors a myriad of genomic and epigenetic defects. Cytosine methylation of CpG-rich promoter DNA is an important mechanism of epigenetic gene inactivation in PCa. There is considerable amount of data to suggest that DNA methylation-based biomarkers may be useful for the early detection and diagnosis of PCa. In addition, candidate gene-based studies have shown an association between specific gene methylation and alterations and clinicopathologic indicators of poor prognosis in PCa.To more comprehensively identify DNA methylation alterations in PCa initiation and progression, we examined the methylation status of 485 577 CpG sites from regions with a broad spectrum of CpG densities, interrogating both gene-associated and non-associated regions using the recently developed Illumina 450K methylation platform.In all, we selected 33 promoter-associated novel CpG sites that were differentially methylated in high-grade prostatic intraepithelial neoplasia and PCa in comparison with benign prostate tissue samples (false discovery rate-adjusted P-value0.05; β-value 0.2; fold change1.5). Of the 33 genes, hierarchical clustering analysis demonstrated BNC1, FZD1, RPL39L, SYN2, LMX1B, CXXC5, ZNF783 and CYB5R2 as top candidate novel genes that are frequently methylated and whose methylation was associated with inactivation of gene expression in PCa cell lines. Pathway analysis of the genes with altered methylation patterns identified the involvement of a cancer-related network of genes whose activity may be regulated by TP53, MYC, TNF, IL1 and 6, IFN-γ and FOS in prostate pathogenesis.Our genome-wide methylation profile shows epigenetic dysregulation of important regulatory signals in prostate carcinogenesis.

Sprouty1 (Spry1) is a negative regulator of fibroblast growth factor signaling with a potential tumor suppressor function in prostate cancer (PCa). Spry1 is downregulated in human PCa, and Spry1 expression can markedly inhibit PCa proliferation in vitro. We have reported DNA methylation as a mechanism for controlling Spry1 expression. However, promoter methylation does not seem to explain gene silencing in all PCa cases studied to suggest other mechanisms of gene inactivation, such as alterations in trans-acting factors and/or post-transcriptional activity may be responsible for the decreased expression in those cases. Binding sites for Wilm's tumor (WT1) transcription factors EGR1, EGR3 and WTE are highly conserved between the mouse and human Spry1 promoter regions, suggesting an evolutionary conserved mechanism(s) involving WT1 and EGR in Spry1 regulation. Spry1 mRNA contains multiple microRNA (miRNA) binding sites in its 3'UTR region suggesting post-transcriptional control. We demonstrate that Spry1 is a target for miR-21-mediated gene silencing. miRNA-based therapeutic approaches to treat cancer are emerging. Spry1 is highly regulated by miRNAs and could potentially be an excellent candidate for such approaches.