Discovering Chromatin Motifs using FAIRE Sequencing and the Human Diploid Genome

Background: Specific chromatin structures are associated with active or inactive gene transcription. The gene regulatory elements are intrinsically dynamic and alternate between inactive and active states through the recruitment of DNA binding proteins, such as chromatin-remodeling proteins. Results: We developed a unique genome-wide method to discover DNA motifs associated with chromatin accessibility using formaldehyde-assisted isolation of regulatory elements with high-throughput sequencing (FAIRE-seq). We aligned the FAIRE-seq reads to the GM12878 diploid genome and subsequently identified differential chromatin-state regions (DCSRs) using heterozygous SNPs. The DCSR pairs represent the locations of imbalances of chromatin accessibility between alleles and are ideal to reveal chromatin motifs that may directly modulate chromatin accessibility. In this study, we used DNA 6-10mer sequences to interrogate all DCSRs, and subsequently discovered conserved chromatin motifs with significant changes in the occurrence frequency. To investigate their likely roles in biology, we studied the annotated protein associated with each of the top ten chromatin motifs genome-wide, in the intergenic regions and in genes, respectively. As a result, we found that most of these annotated motifs are associated with chromatin remodeling, reflecting their significance in biology. Conclusions: Our method is the first one using fully phased diploid genome and FAIRE-seq to discover motifs associated with chromatin accessibility. Our results were collected to construct the first chromatin motif database (CMD), providing the potential DNA motifs recognized by chromatin-remodeling proteins and is freely available at http://syslab.nchu.edu.tw/chromatin

Comparison of visual outcomes after epiretinal membrane surgery

AbstractPurposeTo elucidate the anatomical and visual outcomes of patients with idiopathic epiretinal membranes (ERM) who underwent vitrectomy, membrane removal only, or with internal limiting membrane (ILM) peeling under the assistance of different dyes.MethodsA retrospective chart review of patients with idiopathic ERM who received surgical treatment between January 2004 and December 2009. The patients were grouped according to the usage of staining materials assisting ILM peeling. Group 1 consisted of 61 eyes that underwent conventional vitrectomy and ERM peeling without staining-assisted ILM peeling. Group 2 consisted of 20 eyes with triamcinolone acetonide-assisted ILM peeling following conventional vitrectomy. Group 3 consisted of 23 eyes with indocyanine green-assisted ILM peeling following conventional vitrectomy.ResultsThis study included 104 eyes from 104 patients. There was no significant difference in age, sex, preoperative visual acuity, retinal thickness or follow-up duration among the three groups. Overall, the mean best-corrected visual acuity improved significantly from baseline 0.15 to postoperative 0.41 (p<0.0001). Among the three groups, the mean logarithm minimum angle of resolution acuity markedly improved. There was no significant difference in postoperative visual acuity among groups. As measured by ocular coherent tomography, the mean central foveal thickness decreased from 465.21±86.18 to 299.16±70.14μm. Although there was no difference between groups, postoperative retinal thickness was thicker than that observed in the normal population. The incidence of recurrent ERM was 13.1% in Group 1 and 0% in Groups 2 and 3; this incidence was significantly higher than in the conventional surgery group. Visual outcome was statistically more deteriorated in recurrent cases than in non-recurrent cases (p=0.011).ConclusionsERM surgeries with or without dye-assisted ILM peeling showed similar results. Moreover, the incidence of recurrence is lower in the ILM peeling groups and plays a primary role in determining the final postoperative vision outcome

Hesperetin-7,3'-O-dimethylether selectively inhibits phosphodiesterase 4 and effectively suppresses ovalbumin-induced airway hyperresponsiveness with a high therapeutic ratio

<p>Abstract</p> <p>Background</p> <p>Hesperetin was reported to selectively inhibit phosphodiesterase 4 (PDE4). While hesperetin-7,3'-<it>O</it>-dimethylether (HDME) is a synthetic liposoluble hesperetin. Therefore, we were interested in investigating its selectivity on PDE4 and binding ability on high-affinity rolipram-binding sites (HARBs) <it>in vitro</it>, and its effects on ovalbumin-induced airway hyperresponsiveness <it>in vivo</it>, and clarifying its potential for treating asthma and chronic obstructive pulmonary disease (COPD).</p> <p>Methods</p> <p>PDE1~5 activities were measured using a two-step procedure. The binding of HDME on high-affinity rolipram-binding sites was determined by replacing 2 nM [³<it>H</it>]-rolipram. AHR was assessed using the FlexiVent system and barometric plethysmography. Inflammatory cells were counted using a hemocytometer. Cytokines were determined using mouse T helper (Th)1/Th2 cytokine CBA kits, and total immunoglobulin (Ig)E or IgG_2alevels were done using ELISA method. Xylazine (10 mg/kg)/ketamine (70 mg/kg)-induced anesthesia was performed.</p> <p>Results</p> <p>HDME revealed selective phosphodiesterase 4 (PDE4) inhibition with a therapeutic (PDE4_H/PDE4_L) ratio of 35.5 <it>in vitro</it>. <it>In vivo</it>, HDME (3~30 μmol/kg, orally (p.o.)) dose-dependently and significantly attenuated the airway resistance (R_L) and increased lung dynamic compliance (C_dyn), and decreased enhanced pause (P_enh) values induced by methacholine in sensitized and challenged mice. It also significantly suppressed the increases in the numbers of total inflammatory cells, macrophages, lymphocytes, neutrophils, and eosinophils, and levels of cytokines, including interleukin (IL)-2, IL-4, IL-5, interferon-γ, and tumor necrosis factor-α in bronchoalveolar lavage fluid (BALF) of these mice. In addition, HDME (3~30 μmol/kg, p.o.) dose-dependently and significantly suppressed total and ovalbumin-specific immunoglobulin (Ig)E levels in the BALF and serum, and enhanced IgG_2alevel in the serum of these mice.</p> <p>Conclusions</p> <p>HDME exerted anti-inflammatory effects, including suppression of AHR, and reduced expressions of inflammatory cells and cytokines in this murine model, which appears to be suitable for studying the effects of drugs on atypical asthma and COPD, and for screening those on typical asthma. However, HDME did not influnce xylazine/ketamine-induced anesthesia. Thus HDME may have the potential for use in treating typical and atypical asthma, and COPD.</p

Selection of DDX5 as a novel internal control for Q-RT-PCR from microarray data using a block bootstrap re-sampling scheme

<p>Abstract</p> <p>Background</p> <p>The development of microarrays permits us to monitor transcriptomes on a genome-wide scale. To validate microarray measurements, quantitative-real time-reverse transcription PCR (Q-RT-PCR) is one of the most robust and commonly used approaches. The new challenge in gene quantification analysis is how to explicitly incorporate statistical estimation in such studies. In the realm of statistical analysis, the various available methods of the probe level normalization for microarray analysis may result in distinctly different target selections and variation in the scores for the correlation between microarray and Q-RT-PCR. Moreover, it remains a major challenge to identify a proper internal control for Q-RT-PCR when confirming microarray measurements.</p> <p>Results</p> <p>Sixty-six Affymetrix microarray slides using lung adenocarcinoma tissue RNAs were analyzed by a statistical re-sampling method in order to detect genes with minimal variation in gene expression. By this approach, we identified <it>DDX5 </it>as a novel internal control for Q-RT-PCR. Twenty-three genes, which were differentially expressed between adjacent normal and tumor samples, were selected and analyzed using 24 paired lung adenocarcinoma samples by Q-RT-PCR using two internal controls, <it>DDX5 </it>and <it>GAPDH</it>. The percentage correlation between Q-RT-PCR and microarray were 70% and 48% by using <it>DDX5 </it>and <it>GAPDH </it>as internal controls, respectively.</p> <p>Conclusion</p> <p>Together, these quantification strategies for Q-RT-PCR data processing procedure, which focused on minimal variation, ought to significantly facilitate internal control evaluation and selection for Q-RT-PCR when corroborating microarray data.</p

Butylidenephthalide Blocks Potassium Channels and Enhances Basal Tension in Isolated Guinea-Pig Trachea

Butylidenephthalide (Bdph, 30∼300 M), a constituent of Ligusticum chuanxiong Hort., significantly enhanced tension in isolated guinea-pig trachea. In this study, we investigate the mechanism(s) of Bdph-induced contraction in the tissue. Isolated trachea was bathed in 5 mL of Krebs solution containing indomethacin (3 M), and its tension changes were isometrically recorded. Cromakalim (3 M), an ATP-dependent K + channel opener, significantly antagonized the Bdph-induced enhancement of baseline tension. Bdph (300 M) also significantly antagonized cromakalim-induced relaxation. Bdph (300 M) did not significantly influence the antagonistic effects of glibenclamide (GBC, 1 M) and tetraethylammonium (TEA, 8 mM) against the cromakaliminduced relaxation. However, Bdph (300 M) and 4-aminopiridine (4-AP, 5 mM), a blocker of K 1 family of K + channels, in combination significantly rightward shifted the log concentration-relaxation curve of cromakalim. The antagonistic effect of the combination almost equals the sum of the individual effects of Bdph and 4-AP, suggesting that the antagonistic mechanism of Bdph may be similar to that of 4-AP. All calcium channel blockers influenced neither the baseline tension nor antagonistic effect of Bdph against cromakalim. In conclusion, Bdph may be similar to 4-AP, a blocker of K 1 family of K + channels, to enhance the baseline tension of guinea-pig trachea

Seroprevalence of enterovirus 71 and no evidence of crossprotection of enterovirus 71 antibody against the other enteroviruses in kindergarten children in Taipei city

Background/PurposeEnterovirus 71 (EV71) infection may cause severe neurological and cardiopulmonary complications, especially in preschool children. This study is to investigate the seroprevalence and seroconversion of EV71, and the crossprotection of EV71 antibody against other enteroviruses among kindergarteners.MethodsOverall 228 children in a public kindergarten were enrolled during two academic years, 2006 and 2007, in Taipei, Taiwan and we measured their EV71 neutralizing antibody. When the participants had herpangina; hand, foot and mouth disease (HFMD); febrile illness or respiratory symptoms, throat swabs were sampled and processed for viral culture and enterovirus real-time reverse transcriptase polymerase chain reaction (RT-PCR). Questionnaires, completed by the participants’ guardians, surveyed the history of allergy and annual incidence of symptoms related to enterovirus infection.ResultsSeropositive rates of EV71 were 20% (32/163) in 2006 and 6% (4/65) in 2007. The rate of EV71 seropositivity increased with age (p < 0.01) in 2006 but it did not differ between genders (p = 0.14). No seroconversion was observed from 2006 to 2007. Herpangina occurred in 64% of children with EV71 seropositivity and 48% of those without EV71 antibodies (p = 0.12). Non-71 enterovirus infection, confirmed by viral study, occurred in 53% (19/36) of the EV71-seropositive children and in 53% (102/192) of EV71-seronegative children (p = 0.89). No participants had EV71 infection during the study period.ConclusionEV71 did not frequently circulate in Taipei City from September 2006 to June 2008. Presence of EV71 neutralizing antibody was not associated with lower incidence of enterovirus infection caused by non-71 serotypes

Serologic Status for Pandemic (H1N1) 2009 Virus, Taiwan

We studied preexisting immunity to pandemic (H1N1) 2009 virus in persons in Taiwan. A total of 18 (36%) of 50 elderly adults in Taiwan born before 1935 had protective antibodies against currently circulating pandemic (H1N1) 2009 virus. Seasonal influenza vaccines induced antibodies that did not protect against pandemic (H1N1) 2009 virus

Afatinib Exerts Immunomodulatory Effects by Targeting the Pyrimidine Biosynthesis Enzyme CAD

13 páginas, 7 figurasCurrent clinical trials of combined EGFR-tyrosine kinase inhibitors (TKI) and immune checkpoint blockade (ICB) therapies show no additional effect. This raises questions regarding whether EGFR-TKIs attenuate ICB-enhanced CD8+ T lymphocyte function. Here we show that the EGFR-TKI afatinib suppresses CD8+ T lymphocyte proliferation, and we identify CAD, a key enzyme of de novo pyrimidine biosynthesis, to be a novel afatinib target. Afatinib reduced tumor-infiltrating lymphocyte numbers in Lewis lung carcinoma (LLC)-bearing mice. Early afatinib treatment inhibited CD8+ T lymphocyte proliferation in patients with non-small cell lung cancer, but their proliferation unexpectedly rebounded following long-term treatment. This suggests a transient immunomodulatory effect of afatinib on CD8+ T lymphocytes. Sequential treatment of afatinib with anti-PD1 immunotherapy substantially enhanced therapeutic efficacy in MC38 and LLC-bearing mice, while simultaneous combination therapy showed only marginal improvement over each single treatment. These results suggest that afatinib can suppress CD8+ T lymphocyte proliferation by targeting CAD, proposing a timing window for combined therapy that may prevent the dampening of ICB efficacy by EGFR-TKIs. SIGNIFICANCE: This study elucidates a mechanism of afatinib-mediated immunosuppression and provides new insights into treatment timing for combined targeted therapy and immunotherapy. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/12/3270/F1.large.jpg.This study was supported by Taiwan Ministry of Science and Technology grants MOST 104-2320-B-002-044-MY3, MOST 106-2320-B-002-046-MY3, and MOST 108-2320-B-002-024-MY3, National Health Research Institutes grants NHRI-EX106-10401BI and NHRI-EX109-10725BI, National Taiwan University grants NTU107L890504 and NTU110L893503 to M.-S. Lee, and National Taiwan University Hospital grants 106-003451, 107-003849, 108-004269, and 109-004720 to C.-C. Ho. This work was also supported by MINECO grants BFU2016-80570-R and RTI2018-098084-B-I00 (AEI/FEDER, UE). The authors would like to thank the Laboratory Animal Core Facility at the College of Medicine, National Taiwan University for their servicesPeer reviewe

Pilot Scale Production of Highly Efficacious and Stable Enterovirus 71 Vaccine Candidates

BACKGROUND: Enterovirus 71 (EV71) has caused several epidemics of hand, foot and mouth diseases (HFMD) in Asia and now is being recognized as an important neurotropic virus. Effective medications and prophylactic vaccine against EV71 infection are urgently needed. Based on the success of inactivated poliovirus vaccine, a prototype chemically inactivated EV71 vaccine candidate has been developed and currently in human phase 1 clinical trial. PRINCIPAL FINDING: In this report, we present the development of a serum-free cell-based EV71 vaccine. The optimization at each step of the manufacturing process was investigated, characterized and quantified. In the up-stream process development, different commercially available cell culture media either containing serum or serum-free was screened for cell growth and virus yield using the roller-bottle technology. VP-SFM serum-free medium was selected based on the Vero cell growth profile and EV71 virus production. After the up-stream processes (virus harvest, diafiltration and concentration), a combination of gel-filtration liquid chromatography and/or sucrose-gradient ultracentrifugation down-stream purification processes were investigated at a pilot scale of 40 liters each. Although the combination of chromatography and sucrose-gradient ultracentrifugation produced extremely pure EV71 infectious virus particles, the overall yield of vaccine was 7-10% as determined by a VP2-based quantitative ELISA. Using chromatography as the downstream purification, the virus yield was 30-43%. To retain the integrity of virus neutralization epitopes and the stability of the vaccine product, the best virus inactivation was found to be 0.025% formalin-treatment at 37 °C for 3 to 6 days. Furthermore, the formalin-inactivated virion vaccine candidate was found to be stable for >18 months at 4 °C and a microgram of viral proteins formulated with alum adjuvant could induce strong virus-neutralizing antibody responses in mice, rats, rabbits, and non-human primates. CONCLUSION: These results provide valuable information supporting the current cell-based serum-free EV71 vaccine candidate going into human Phase I clinical trials