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1.  The expanding territories of condensin II 
Cell Cycle  2015;14(17):2723-2724.
doi:10.1080/15384101.2015.1063356
PMCID: PMC4614856  PMID: 26280196
2.  Long‐Term Prognosis of Brugada‐Type ECG and ECG With Atypical ST‐Segment Elevation in the Right Precordial Leads Over 20 Years: Results From the Circulatory Risk in Communities Study (CIRCS) 
Background
Brugada syndrome is recognized as being associated with sudden cardiac death; however, the prevalence of non–type 1 Brugada‐type ECG (BrS) or atypical ST‐segment elevation in the right precordial leads (STERP) and the long‐term prognosis for those patients remain unknown.
Methods and Results
We analyzed standard 12‐lead ECGs of 7178 apparently healthy participants (age range 40–64 years) who underwent health checkups from 1982 to 1986 in the Circulatory Risk in Communities Study, a prospective, large, community‐based cohort study in Japan. ECGs with J point amplitude ≥0.2 mV in the right precordial leads were divided into 3 groups: (1) type 1 BrS, (2) type 2 or 3 BrS (non‐type 1 BrS), and (3) STERP. The others served as the non–ST‐segment elevation group. We identified 8 participants (0.1%) with type1 BrS, 84 (1.2%) with non–type 1 BrS, and 228 (3.2%) with STERP. During a median follow‐up of 18.7 years (133 987.0 person‐years), sudden cardiac death was observed in no participants (0.0%) with type 1 BrS, in 1 (1.2%) with non–type 1 BrS, in 7 (3.1%) with STERP, and in 50 (0.7%) with non–ST‐segment elevation. Participants with STERP had a markedly elevated risk of sudden cardiac death (multivariable hazard ratio 3.9, 95% CI 1.7–9.0).
Conclusions
STERP was associated with an elevated risk of sudden cardiac death in a middle‐aged population.
doi:10.1161/JAHA.115.002899
PMCID: PMC5015268  PMID: 27503848
Brugada syndrome; electrocardiography; epidemiology; prognosis; sudden cardiac death; Sudden Cardiac Death
4.  Transcriptional silencing of long noncoding RNA GNG12-AS1 uncouples its transcriptional and product-related functions 
Nature Communications  2016;7:10406.
Long noncoding RNAs (lncRNAs) regulate gene expression via their RNA product or through transcriptional interference, yet a strategy to differentiate these two processes is lacking. To address this, we used multiple small interfering RNAs (siRNAs) to silence GNG12-AS1, a nuclear lncRNA transcribed in an antisense orientation to the tumour-suppressor DIRAS3. Here we show that while most siRNAs silence GNG12-AS1 post-transcriptionally, siRNA complementary to exon 1 of GNG12-AS1 suppresses its transcription by recruiting Argonaute 2 and inhibiting RNA polymerase II binding. Transcriptional, but not post-transcriptional, silencing of GNG12-AS1 causes concomitant upregulation of DIRAS3, indicating a function in transcriptional interference. This change in DIRAS3 expression is sufficient to impair cell cycle progression. In addition, the reduction in GNG12-AS1 transcripts alters MET signalling and cell migration, but these are independent of DIRAS3. Thus, differential siRNA targeting of a lncRNA allows dissection of the functions related to the process and products of its transcription.
LncRNAs regulate gene expression via their RNA product and through transcriptional interference. Here, Stojic et al. uncouple these functions by using multiple siRNAs against GNG12-AS1 to show that this lncRNA has a product related role in MET signaling while its transcription modulates DIRAS3 expression.
doi:10.1038/ncomms10406
PMCID: PMC4740813  PMID: 26832224
5.  Stanniocalcin-1 is induced by Hypoxia Inducible Factor in Rat Alveolar Epithelial Cells 
Alveolar type II (ATII) cells remain differentiated and express surfactant proteins when cultured at an air-liquid (A/L) interface. When cultured under submerged conditions, ATII cells dedifferentiate and change their gene expression profile. We have previously shown that gene expression under submerged conditions is regulated by hypoxia inducible factor (HIF) signaling due to focal hypoxia resulting from ATII cell metabolism. Herein, we sought to further define gene expression changes in ATII cells cultured under submerged conditions. We performed a genome wide microarray on RNA extracted from rat ATII cells cultured under submerged conditions for 24–48 h after switching from an A/L interface. We found significant alterations in gene expression, including upregulation of the HIF target genes stanniocalcin-1 (STC1), tyrosine hydroxylase (Th), enolase (Eno) 2, and matrix metalloproteinase (MMP) 13, and we verified upregulation of these genes by RT-PCR. Because STC1, a highly evolutionarily conserved glycoprotein with anti-inflammatory, anti-apoptotic, anti-oxidant, and wound healing properties, is widely expressed in the lung, we further explored the potential functions of STC1 in the alveolar epithelium. We found that STC1 was induced by hypoxia and HIF in rat ATII cells, and this induction occurred rapidly and reversibly. We also showed that recombinant human STC1 (rhSTC1) enhanced cell motility with extended lamellipodia formation in alveolar epithelial cell (AEC) monolayers but did not inhibit the oxidative damage induced by LPS. We also confirmed that STC1 was upregulated by hypoxia and HIF in human lung epithelial cells. In this study, we have found that several HIF target genes including STC1 are upregulated in AECs by a submerged condition, that STC1 is regulated by hypoxia and HIF, that this regulation is rapidly and reversibly, and that STC1 enhances wound healing moderately in AEC monolayers. However, STC1 did not inhibit oxidative damage in rat AECs stimulated by LPS in vitro. Therefore, alterations in gene expression by ATII cells under submerged conditions including STC1 were largely induced by hypoxia and HIF, which may be relevant to our understanding of the pathogenesis of various lung diseases in which the alveolar epithelium is exposed to relative hypoxia.
doi:10.1016/j.bbrc.2014.09.060
PMCID: PMC4188510  PMID: 25251473
HIF; STC1; Gene expression; Alveolar epithelial cells
6.  Ability of magnetocardiography to detect regional dominant frequencies of atrial fibrillation 
Journal of Arrhythmia  2015;31(6):345-351.
Background
Lead V1 on electrocardiography (ECG) can detect the dominant frequency (DF) of atrial fibrillation (AF) in the right atrium (RA). Paroxysmal AF is characterized by a frequency gradient from the left atrium (LA) to the right atrium (RA). We examined the ability of magnetocardiography (MCG) to detect regional DFs in both the atria.
Methods
Study subjects comprised 18 consecutive patients referred for catheter ablation of persistent AF. An MCG system with 64 magnetic sensors was used to perform MCG in the frontal, lateral, and back planes prior to the ablation procedure in each patient. DFMCG and organization index (OIMCG) were calculated using fast Fourier transformation. Intracardiac electrograms (ICEs) in both the atria and the coronary sinus (CS) were mapped at 17 sites. Regional DFsICE were also determined.
Results
Mean LA DFICE was higher than mean RA DFICE (6.40±0.66 versus 6.16±0.80 Hz, P=0.03). DFMCG in the channel having the highest OIMCG was 6.61±0.88 Hz in the frontal plane, 6.52±0.64 Hz in the lateral plane, and 6.42±0.62 Hz in the back plane (P=0.3). In each plane, DFMCG correlated with DFICE at the RA appendage (R=0.95, P<0.0001), the LA appendage (R=0.91, P<0.0001), and the CS (R=0.93, P<0.0001). DFECG in V5 modestly correlated with DFICE at the LA appendage (R=0.82, P<0.0001).
Conclusions
MCG could more precisely detect the DFs in the LA and the CS than ECG. However, the usefulness of pre-procedural detection of the AF frequency gradient for ablation therapy needs to be evaluated in future prospective studies.
doi:10.1016/j.joa.2015.05.003
PMCID: PMC4672036  PMID: 26702313
Atrial fibrillation; Magnetocardiography; Dominant frequency
7.  Influenza induces IL-8 and GM-CSF secretion by human alveolar epithelial cells through HGF/c-Met and TGF-α/EGFR signaling 
The most severe complication of influenza is viral pneumonia, which can lead to the acute respiratory distress syndrome. Alveolar epithelial cells (AECs) are the first cells that influenza virus encounters upon entering the alveolus. Infected epithelial cells produce cytokines that attract and activate neutrophils and macrophages, which in turn induce damage to the epithelial-endothelial barrier. Hepatocyte growth factor (HGF)/c-Met and transforming growth factor-α (TGF-α)/epidermal growth factor receptor (EGFR) are well known to regulate repair of damaged alveolar epithelium by stimulating cell migration and proliferation. Recently, TGF-α/EGFR signaling has also been shown to regulate innate immune responses in bronchial epithelial cells. However, little is known about whether HGF/c-Met signaling alters the innate immune responses and whether the innate immune responses in AECs are regulated by HGF/c-Met and TGF-α/EGFR. We hypothesized that HGF/c-Met and TGF-α/EGFR would regulate innate immune responses to influenza A virus infection in human AECs. We found that recombinant human HGF (rhHGF) and rhTGF-α stimulated primary human AECs to secrete IL-8 and granulocyte macrophage colony-stimulating factor (GM-CSF) strongly and IL-6 and monocyte chemotactic protein 1 moderately. Influenza infection stimulated the secretion of IL-8 and GM-CSF by AECs plated on rat-tail collagen through EGFR activation likely by TGF-α released from AECs and through c-Met activated by HGF secreted from lung fibroblasts. HGF secretion by fibroblasts was stimulated by AEC production of prostaglandin E2 during influenza infection. We conclude that HGF/c-Met and TGF-α/EGFR signaling enhances the innate immune responses by human AECs during influenza infections.
doi:10.1152/ajplung.00290.2014
PMCID: PMC4451400  PMID: 26033355
alveolar epithelial cells; influenza; innate immune responses; hepatocyte growth factor/c-Met; transforming growth factor-α/epidermal growth factor receptor
8.  5-hydroxymethylcytosine marks promoters in colon that resist DNA hypermethylation in cancer 
Genome Biology  2015;16(1):69.
Background
The discovery of cytosine hydroxymethylation (5hmC) as a mechanism that potentially controls DNA methylation changes typical of neoplasia prompted us to investigate its behaviour in colon cancer. 5hmC is globally reduced in proliferating cells such as colon tumours and the gut crypt progenitors, from which tumours can arise.
Results
Here, we show that colorectal tumours and cancer cells express Ten-Eleven-Translocation (TET) transcripts at levels similar to normal tissues. Genome-wide analyses show that promoters marked by 5hmC in normal tissue, and those identified as TET2 targets in colorectal cancer cells, are resistant to methylation gain in cancer. In vitro studies of TET2 in cancer cells confirm that these promoters are resistant to methylation gain independently of sustained TET2 expression. We also find that a considerable number of the methylation gain-resistant promoters marked by 5hmC in normal colon overlap with those that are marked with poised bivalent histone modifications in embryonic stem cells.
Conclusions
Together our results indicate that promoters that acquire 5hmC upon normal colon differentiation are innately resistant to neoplastic hypermethylation by mechanisms that do not require high levels of 5hmC in tumours. Our study highlights the potential of cytosine modifications as biomarkers of cancerous cell proliferation.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-015-0605-5) contains supplementary material, which is available to authorized users.
doi:10.1186/s13059-015-0605-5
PMCID: PMC4380107  PMID: 25853800
9.  Phenotype Specific Analyses Reveal Distinct Regulatory Mechanism for Chronically Activated p53 
PLoS Genetics  2015;11(3):e1005053.
The downstream functions of the DNA binding tumor suppressor p53 vary depending on the cellular context, and persistent p53 activation has recently been implicated in tumor suppression and senescence. However, genome-wide information about p53-target gene regulation has been derived mostly from acute genotoxic conditions. Using ChIP-seq and expression data, we have found distinct p53 binding profiles between acutely activated (through DNA damage) and chronically activated (in senescent or pro-apoptotic conditions) p53. Compared to the classical ‘acute’ p53 binding profile, ‘chronic’ p53 peaks were closely associated with CpG-islands. Furthermore, the chronic CpG-island binding of p53 conferred distinct expression patterns between senescent and pro-apoptotic conditions. Using the p53 targets seen in the chronic conditions together with external high-throughput datasets, we have built p53 networks that revealed extensive self-regulatory ‘p53 hubs’ where p53 and many p53 targets can physically interact with each other. Integrating these results with public clinical datasets identified the cancer-associated lipogenic enzyme, SCD, which we found to be directly repressed by p53 through the CpG-island promoter, providing a mechanistic link between p53 and the ‘lipogenic phenotype’, a hallmark of cancer. Our data reveal distinct phenotype associations of chronic p53 targets that underlie specific gene regulatory mechanisms.
Author Summary
The p53 transcription factor is a frequently mutated tumour suppressor that contributes to repairing or eliminating damaged cells. Levels of p53 are typically regulated through its stability; it is constantly produced and degraded, so that upon stress, p53 is up-regulated quickly. This acutely induced p53 has been used as a major model system for studying genome-wide p53 targets. However, emerging evidence suggests that persistently activated p53 is involved in cancer-associated phenotypes, such as cellular senescence. We investigate genome-wide gene regulation by acutely induced p53 through DNA damage as well as chronically activated p53 in oncogene-induced senescence and pro-apoptotic states. Interestingly, acute and chronic p53 DNA binding profiles are highly distinctive, the latter being preferentially associated with larger and relatively open promoters called CpG islands. Furthermore, our integrative analyses of both p53-dependent gene expression and p53-binding genomic DNA profiles reveal that p53 and many of its targets in chronic conditions form extensive self-regulatory hubs, where they can physically interact. The data not only substantially extend the list of direct p53 targets but highlight unique gene regulation by chronic p53. Finally we show that the cancer-associated lipogenic enzyme, stearoyl-CoA desaturase, is a bona fide p53-repressive target through its CpG island promoter in chronic conditions.
doi:10.1371/journal.pgen.1005053
PMCID: PMC4366240  PMID: 25790137
10.  Innate Immune Response of Human Alveolar Type II Cells Infected with Severe Acute Respiratory Syndrome–Coronavirus 
Severe acute respiratory syndrome (SARS)–coronavirus (CoV) produces a devastating primary viral pneumonia with diffuse alveolar damage and a marked increase in circulating cytokines. One of the major cell types to be infected is the alveolar type II cell. However, the innate immune response of primary human alveolar epithelial cells infected with SARS-CoV has not been defined. Our objectives included developing a culture system permissive for SARS-CoV infection in primary human type II cells and defining their innate immune response. Culturing primary human alveolar type II cells at an air–liquid interface (A/L) improved their differentiation and greatly increased their susceptibility to infection, allowing us to define their primary interferon and chemokine responses. Viral antigens were detected in the cytoplasm of infected type II cells, electron micrographs demonstrated secretory vesicles filled with virions, virus RNA concentrations increased with time, and infectious virions were released by exocytosis from the apical surface of polarized type II cells. A marked increase was evident in the mRNA concentrations of interferon–β and interferon–λ (IL-29) and in a large number of proinflammatory cytokines and chemokines. A surprising finding involved the variability of expression of angiotensin-converting enzyme–2, the SARS-CoV receptor, in type II cells from different donors. In conclusion, the cultivation of alveolar type II cells at an air–liquid interface provides primary cultures in which to study the pulmonary innate immune responses to infection with SARS-CoV, and to explore possible therapeutic approaches to modulating these innate immune responses.
doi:10.1165/rcmb.2012-0339OC
PMCID: PMC3727876  PMID: 23418343
lung innate immune response; cytokine responses to SARS coronavirus; lung cell differentiation; air–liquid interface cultures
11.  MicroRNA-196a Is a Putative Diagnostic Biomarker and Therapeutic Target for Laryngeal Cancer 
PLoS ONE  2013;8(8):e71480.
Background
MicroRNA (miRNA) is an emerging subclass of small non-coding RNAs that regulates gene expression and has a pivotal role for many physiological processes including cancer development. Recent reports revealed the role of miRNAs as ideal biomarkers and therapeutic targets due to their tissue- or disease-specific nature. Head and neck cancer (HNC) is a major cause of cancer-related mortality and morbidity, and laryngeal cancer has the highest incidence in it. However, the molecular mechanisms involved in laryngeal cancer development remain to be known and highly sensitive biomarkers and novel promising therapy is necessary.
Methodology/Principal Findings
To explore laryngeal cancer-specific miRNAs, RNA from 5 laryngeal surgical specimens including cancer and non-cancer tissues were hybridized to microarray carrying 723 human miRNAs. The resultant differentially expressed miRNAs were further tested by using quantitative real time PCR (qRT-PCR) on 43 laryngeal tissue samples including cancers, noncancerous counterparts, benign diseases and precancerous dysplasias. Significant expressional differences between matched pairs were reproduced in miR-133b, miR-455-5p, and miR-196a, among which miR-196a being the most promising cancer biomarker as validated by qRT-PCR analyses on additional 84 tissue samples. Deep sequencing analysis revealed both quantitative and qualitative deviation of miR-196a isomiR expression in laryngeal cancer. In situ hybridization confirmed laryngeal cancer-specific expression of miR-196a in both cancer and cancer stroma cells. Finally, inhibition of miR-196a counteracted cancer cell proliferation in both laryngeal cancer-derived cells and mouse xenograft model.
Conclusions/Significance
Our study provided the possibilities that miR-196a might be very useful in diagnosing and treating laryngeal cancer.
doi:10.1371/journal.pone.0071480
PMCID: PMC3743786  PMID: 23967217
12.  A Novel Mechanism for CTCF in the Epigenetic Regulation of Bax in Breast Cancer Cells12 
Neoplasia (New York, N.Y.)  2013;15(8):898-912.
We previously reported the association of elevated levels of the multifunctional transcription factor, CCCTC binding factor (CTCF), in breast cancer cells with the specific anti-apoptotic function of CTCF. To understand the molecular mechanisms of this phenomenon, we investigated regulation of the human Bax gene by CTCF in breast and non-breast cells. Two CTCF binding sites (CTSs) within the Bax promoter were identified. In all cells, breast and non-breast, active histone modifications were present at these CTSs, DNA harboring this region was unmethylated, and levels of Bax mRNA and protein were similar. Nevertheless, up-regulation of Bax mRNA and protein and apoptotic cell death were observed only in breast cancer cells depleted of CTCF. We proposed that increased CTCF binding to the Bax promoter in breast cancer cells, by comparison with non-breast cells, may be mechanistically linked to the specific apoptotic phenotype in CTCF-depleted breast cancer cells. In this study, we show that CTCF binding was enriched at the Bax CTSs in breast cancer cells and tumors; in contrast, binding of other transcription factors (SP1, WT1, EGR1, and c-Myc) was generally increased in non-breast cells and normal breast tissues. Our findings suggest a novel mechanism for CTCF in the epigenetic regulation of Bax in breast cancer cells, whereby elevated levels of CTCF support preferential binding of CTCF to the Bax CTSs. In this context, CTCF functions as a transcriptional repressor counteracting influences of positive regulatory factors; depletion of breast cancer cells from CTCF therefore results in the activation of Bax and apoptosis.
PMCID: PMC3730042  PMID: 23908591
13.  Induced DNA demethylation can reshape chromatin topology at the IGF2-H19 locus 
Nucleic Acids Research  2013;41(10):5290-5302.
Choriocarcinomas are embryonal tumours with loss of imprinting and hypermethylation at the insulin-like growth factor 2 (IGF2)-H19 locus. The DNA methyltransferase inhibitor, 5-Aza-2′deoxycytidine (5-AzaCdR) is an approved epigenetic cancer therapy. However, it is not known to what extent 5-AzaCdR influences other epigenetic marks. In this study, we set out to determine whether 5-AzaCdR treatment can reprogram the epigenomic organization of the IGF2-H19 locus in a choriocarcinoma cancer cell line (JEG3). We found that localized DNA demethylation at the H19 imprinting control region (ICR) induced by 5-AzaCdR, reduced IGF2, increased H19 expression, increased CTCF and cohesin recruitment and changed histone modifications. Furthermore chromatin accessibility was increased locus-wide and chromatin looping topography was altered such that a CTCF site downstream of the H19 enhancers switched its association with the CTCF site upstream of the IGF2 promoters to associate with the ICR. We identified a stable chromatin looping domain, which forms independently of DNA methylation. This domain contains the IGF2 gene and is marked by a histone H3 lysine 27 trimethylation block between CTCF site upstream of the IGF2 promoters and the Centrally Conserved Domain upstream of the ICR. Together, these data provide new insights into the responsiveness of chromatin topography to DNA methylation changes.
doi:10.1093/nar/gkt240
PMCID: PMC3664821  PMID: 23585276
14.  Infection of human alveolar macrophages by human coronavirus strain 229E 
The Journal of General Virology  2012;93(Pt 3):494-503.
Human coronavirus strain 229E (HCoV-229E) commonly causes upper respiratory tract infections. However, lower respiratory tract infections can occur in some individuals, indicating that cells in the distal lung are susceptible to HCoV-229E. This study determined the virus susceptibility of primary cultures of human alveolar epithelial cells and alveolar macrophages (AMs). Fluorescent antibody staining indicated that HCoV-229E could readily infect AMs, but no evidence was found for infection in differentiated alveolar epithelial type II cells and only a very low level of infection in type II cells transitioning to the type I-like cell phenotype. However, a human bronchial epithelial cell line (16HBE) was readily infected. The innate immune response of AMs to HCoV-229E infection was evaluated for cytokine production and interferon (IFN) gene expression. AMs secreted significant amounts of tumour necrosis factor alpha (TNF-α), regulated on activation normal T-cell expressed and secreted (RANTES/CCL5) and macrophage inflammatory protein 1β (MIP-1β/CCL4) in response to HCoV-229E infection, but these cells exhibited no detectable increase in IFN-β or interleukin-29 in mRNA levels. AMs from smokers had reduced secretion of TNF-α compared with non-smokers in response to HCoV-229E infection. Surfactant protein A (SP-A) and SP-D are part of the innate immune system in the distal lung. Both surfactant proteins bound to HCoV-229E, and pre-treatment of HCoV-229E with SP-A or SP-D inhibited infection of 16HBE cells. In contrast, there was a modest reduction in infection in AMs by SP-A, but not by SP-D. In summary, AMs are an important target for HCoV-229E, and they can mount a pro-inflammatory innate immune response to infection.
doi:10.1099/vir.0.038414-0
PMCID: PMC3352353  PMID: 22090214
15.  Hypoxia-Inducible Factor Regulates Expression of Surfactant Protein in Alveolar Type II Cells In Vitro 
Alveolar type II (ATII) cells cultured at an air–liquid (A/L) interface maintain differentiation, but they lose these properties when they are submerged. Others showed that an oxygen tension gradient develops in the culture medium as ATII cells consume oxygen. Therefore, we wondered whether hypoxia inducible factor (HIF) signaling could explain differences in the phenotypes of ATII cells cultured under A/L interface or submerged conditions. ATII cells were isolated from male Sprague-Dawley rats and cultured on inserts coated with a mixture of rat-tail collagen and Matrigel, in medium including 5% rat serum and 10 ng/ml keratinocyte growth factor, with their apical surfaces either exposed to air or submerged. The A/L interface condition maintained the expression of surfactant proteins, whereas that expression was down-regulated under the submerged condition, and the effect was rapid and reversible. Under submerged conditions, there was an increase in HIF1α and HIF2α in nuclear extracts, mRNA levels of HIF inducible genes, vascular endothelial growth factor, glucose transporter–1 (GLUT1), and the protein level of pyruvate dehydrogenase kinase isozyme–1. The expression of surfactant proteins was suppressed and GLUT1 mRNA levels were induced when cells were cultured with 1 mM dimethyloxalyl glycine. The expression of surfactant proteins was restored under submerged conditions with supplemented 60% oxygen. HIF signaling and oxygen tension at the surface of cells appears to be important in regulating the phenotype of rat ATII cells.
doi:10.1165/rcmb.2011-0052OC
PMCID: PMC3262688  PMID: 21454802
HIF; ATII cells; surfactant proteins; VEGF; GLUT1
16.  cis-Golgi proteins accumulate near the ER exit sites and act as the scaffold for Golgi regeneration after brefeldin A treatment in tobacco BY-2 cells 
Molecular Biology of the Cell  2012;23(16):3203-3214.
Particular cis-Golgi proteins accumulate in novel punctate structures close to ERES by BFA treatment in tobacco BY-2 cells. These structures reassemble first to form cis-Golgi after BFA removal, and the Golgi stacks regenerate in the cis-to-trans order. This indicates that the punctate structures act as the scaffold for Golgi regeneration.
The Golgi apparatus forms stacks of cisternae in many eukaryotic cells. However, little is known about how such a stacked structure is formed and maintained. To address this question, plant cells provide a system suitable for live-imaging approaches because individual Golgi stacks are well separated in the cytoplasm. We established tobacco BY-2 cell lines expressing multiple Golgi markers tagged by different fluorescent proteins and observed their responses to brefeldin A (BFA) treatment and BFA removal. BFA treatment disrupted cis, medial, and trans cisternae but caused distinct relocalization patterns depending on the proteins examined. Medial- and trans-Golgi proteins, as well as one cis-Golgi protein, were absorbed into the endoplasmic reticulum (ER), but two other cis-Golgi proteins formed small punctate structures. After BFA removal, these puncta coalesced first, and then the Golgi stacks regenerated from them in the cis-to-trans order. We suggest that these structures have a property similar to the ER-Golgi intermediate compartment and function as the scaffold of Golgi regeneration.
doi:10.1091/mbc.E12-01-0034
PMCID: PMC3418314  PMID: 22740633
17.  Chemotherapy-induced complications in patients with lung cancer: An evaluation by pharmacists 
In lung cancer patients, chemotherapy-induced complications are considered to be distressing reactions even in the era of new antiemetics, such as aprepitant. The aim of this study was to evaluate the incidence of such complications. This prospective observational study was performed in our institution between 2011 and 2012. Certain complications including nausea, vomiting, appetite, stomatitis, constipation, diarrhea and dysesthesia, on days 1–7 were evaluated by pharmacists. The questionnaires and diaries of chemotherapy-induced complications were evaluated in the 31 patients included in the study. The majority of the enrolled patients were male (81%). Six (19%) patients were administered cis-diamminedichloroplatinum(II) (CDDP)-, 21 (69%) chemotherapy by carboplatin (CBDCA)- and 4 (13%) non-platinum regimen chemotherapies. Ten (32.3%) of the 31 patients exhibited nausea but only 3 (9.7%) of them experienced vomiting. On days 5–6, 23.8 and 9.5%, respectively, of patients treated with CDDP-regimens had nausea and vomiting. Three of the other most common complications were constipation, general fatigue and appetite loss. The incidence of these complications was 77.4, 71.0 and 67.7%, respectively. Even in the era of new antiemetics, CDDP-regimen chemotherapy-induced nausea and vomiting as well as constipation; general fatigue and appetite loss continue to be problems. A better appreciation of the incidence of these chemotherapy-related complications by medical oncologists and medical staff is essential for their adequate control.
doi:10.3892/mco.2012.33
PMCID: PMC3956253  PMID: 24649124
chemotherapy-induced complications; lung cancer; pharmacists
18.  Innate Immune Response to Influenza A Virus in Differentiated Human Alveolar Type II Cells 
Alveolar Type II (ATII) cells are important targets for seasonal and pandemic influenza. To investigate the influenza-induced innate immune response in those cells, we measured the global gene expression profile of highly differentiated ATII cells infected with the influenza A virus at a multiplicity of infection of 0.5 at 4 hours and 24 hours after inoculation. Infection with influenza stimulated a significant increase in the mRNA concentrations of many host defense–related genes, including pattern/pathogen recognition receptors, IFN, and IFN-induced genes, chemokines, and suppressors of cytokine signaling. We verified these changes by quantitative real-time RT-PCR. At the protein level, we detected a robust virus-induced secretion of the three glutamic acid-leucine-arginine (ELR)-negative chemokines CXCL9, CXCL10, and CXCL11, according to ELISA. The ultraviolet inactivation of virus abolished the chemokine and cytokine response. Viral infection did not appear to alter the differentiation of ATII cells, as measured by cellular mRNA and concentrations of surfactant proteins. However, viral infection significantly reduced the secretion of surfactant protein (SP)–A and SP–D. In addition, influenza A virus triggered a time-dependent activation of phosphatidylinositol 3–kinase signaling in ATII cells. The inhibition of this pathway significantly decreased the release of infectious virus and the chemokine response, but did not alter virus-induced cell death. This study provides insights into influenza-induced innate immunity in differentiated human ATII cells, and demonstrates that the alveolar epithelium is a critical part of the initial innate immune response to influenza.
doi:10.1165/rcmb.2010-0108OC
PMCID: PMC3175576  PMID: 21239608
human Type II cell; influenza; chemokine; PI3k; differentiation
19.  Innate Immune Response of Human Alveolar Macrophages during Influenza A Infection 
PLoS ONE  2012;7(3):e29879.
Alveolar macrophages (AM) are one of the key cell types for initiating inflammatory and immune responses to influenza virus in the lung. However, the genome-wide changes in response to influenza infection in AM have not been defined. We performed gene profiling of human AM in response to H1N1 influenza A virus PR/8 using Affymetrix HG-U133 Plus 2.0 chips and verified the changes at both mRNA and protein levels by real-time RT-PCR and ELISA. We confirmed the response with a contemporary H3N2 influenza virus A/New York/238/2005 (NY/238). To understand the local cellular response, we also evaluated the impact of paracrine factors on virus-induced chemokine and cytokine secretion. In addition, we investigated the changes in the expression of macrophage receptors and uptake of pathogens after PR/8 infection. Although macrophages fail to release a large amount of infectious virus, we observed a robust induction of type I and type III interferons and several cytokines and chemokines following influenza infection. CXCL9, 10, and 11 were the most highly induced chemokines by influenza infection. UV-inactivation abolished virus-induced cytokine and chemokine response, with the exception of CXCL10. The contemporary influenza virus NY/238 infection of AM induced a similar response as PR/8. Inhibition of TNF and/or IL-1β activity significantly decreased the secretion of the proinflammatory chemokines CCL5 and CXCL8 by over 50%. PR/8 infection also significantly decreased mRNA levels of macrophage receptors including C-type lectin domain family 7 member A (CLEC7A), macrophage scavenger receptor 1 (MSR1), and CD36, and reduced uptake of zymosan. In conclusion, influenza infection induced an extensive proinflammatory response in human AM. Targeting local components of innate immune response might provide a strategy for controlling influenza A infection-induced proinflammatory response in vivo.
doi:10.1371/journal.pone.0029879
PMCID: PMC3292548  PMID: 22396727
20.  Disruption of genomic neighbourhood at the imprinted IGF2-H19 locus in Beckwith–Wiedemann syndrome and Silver–Russell syndrome 
Human Molecular Genetics  2011;20(7):1363-1374.
Hyper- and hypomethylation at the IGF2-H19 imprinting control region (ICR) result in reciprocal changes in IGF2-H19 expression and the two contrasting growth disorders, Beckwith–Wiedemann syndrome (BWS) and Silver–Russell syndrome (SRS). DNA methylation of the ICR controls the reciprocal imprinting of IGF2 and H19 by preventing the binding of the insulator protein, CTCF. We here show that local changes in histone modifications and CTCF–cohesin binding at the ICR in BWS and SRS together with DNA methylation correlate with the higher order chromatin structure at the locus. In lymphoblastoid cells from control individuals, we found the repressive histone H3K9me3 and H4K20me3 marks associated with the methylated paternal ICR allele and the bivalent H3K4me2/H3K27me3 mark together with H3K9ac and CTCF–cohesin associated with the non-methylated maternal allele. In patient-derived cell lines, the mat/pat asymmetric distribution of these epigenetic marks was lost with H3K9me3 and H4K20me3 becoming biallelic in the BWS and H3K4me2, H3K27me3 and H3K9ac together with CTCF–cohesin becoming biallelic in the SRS. We further show that in BWS and SRS cells, there is opposing chromatin looping conformation mediated by CTCF–cohesin binding sites surrounding the locus. In normal cells, lack of CTCF–cohesin binding at the paternal ICR is associated with monoallelic interaction between two CTCF sites flanking the locus. CTCF–cohesin binding at the maternal ICR blocks this interaction by associating with the CTCF site downstream of the enhancers. The two alternative chromatin conformations are differently favoured in BWS and SRS likely predisposing the locus to the activation of IGF2 or H19, respectively.
doi:10.1093/hmg/ddr018
PMCID: PMC3049359  PMID: 21282187
21.  Rat respiratory coronavirus infection: replication in airway and alveolar epithelial cells and the innate immune response 
The Journal of General Virology  2009;90(Pt 12):2956-2964.
The rat coronavirus sialodacryoadenitis virus (SDAV) causes respiratory infection and provides a system for investigating respiratory coronaviruses in a natural host. A viral suspension in the form of a microspray aerosol was delivered by intratracheal instillation into the distal lung of 6–8-week-old Fischer 344 rats. SDAV inoculation produced a 7 % body weight loss over a 5 day period that was followed by recovery over the next 7 days. SDAV caused focal lesions in the lung, which were most severe on day 4 post-inoculation (p.i.). Immunofluorescent staining showed that four cell types supported SDAV virus replication in the lower respiratory tract, namely Clara cells, ciliated cells in the bronchial airway and alveolar type I and type II cells in the lung parenchyma. In bronchial alveolar lavage fluid (BALF) a neutrophil influx increased the population of neutrophils to 45 % compared with 6 % of the cells in control samples on day 2 after mock inoculation. Virus infection induced an increase in surfactant protein SP-D levels in BALF of infected rats on days 4 and 8 p.i. that subsided by day 12. The concentrations of chemokines MCP-1, LIX and CINC-1 in BALF increased on day 4 p.i., but returned to control levels by day 8. Intratracheal instillation of rats with SDAV coronavirus caused an acute, self-limited infection that is a useful model for studying the early events of the innate immune response to respiratory coronavirus infections in lungs of the natural virus host.
doi:10.1099/vir.0.014282-0
PMCID: PMC2887555  PMID: 19741068
22.  The effect of interleukin-13 (IL-13) and interferon-γ (IFN-γ) on expression of surfactant proteins in adult human alveolar type II cells in vitro 
Respiratory Research  2010;11(1):157.
Background
Surfactant proteins are produced predominantly by alveolar type II (ATII) cells, and the expression of these proteins can be altered by cytokines and growth factors. Th1/Th2 cytokine imbalance is suggested to be important in the pathogenesis of several adult lung diseases. Recently, we developed a culture system for maintaining differentiated adult human ATII cells. Therefore, we sought to determine the effects of IL-13 and IFN-γ on the expression of surfactant proteins in adult human ATII cells in vitro. Additional studies were done with rat ATII cells.
Methods
Adult human ATII cells were isolated from deidentified organ donors whose lungs were not suitable for transplantation and donated for medical research. The cells were cultured on a mixture of Matrigel and rat-tail collagen for 8 d with differentiation factors and human recombinant IL-13 or IFN-γ.
Results
IL-13 reduced the mRNA and protein levels of surfactant protein (SP)-C, whereas IFN-γ increased the mRNA level of SP-C and proSP-C protein but not mature SP-C. Neither cytokine changed the mRNA level of SP-B but IFN-γ slightly decreased mature SP-B. IFN-γ reduced the level of the active form of cathepsin H. IL-13 also reduced the mRNA and protein levels of SP-D, whereas IFN-γ increased both mRNA and protein levels of SP-D. IL-13 did not alter SP-A, but IFN-γ slightly increased the mRNA levels of SP-A.
Conclusions
We demonstrated that IL-13 and IFN-γ altered the expression of surfactant proteins in human adult ATII cells in vitro. IL-13 decreased SP-C and SP-D in human ATII cells, whereas IFN-γ had the opposite effect. The protein levels of mature SP-B were decreased by IFN-γ treatment, likely due to the reduction in active form cathpesin H. Similarly, the active form of cathepsin H was relatively insufficient to fully process proSP-C as IFN-γ increased the mRNA levels for SP-C and proSP-C protein, but there was no increase in mature SP-C. These observations suggest that in disease states with an overexpression of IL-13, there would be some deficiency in mature SP-C and SP-D. In disease states with an excess of IFN-γ or therapy with IFN-γ, these data suggest that there might be incomplete processing of SP-B and SP-C.
doi:10.1186/1465-9921-11-157
PMCID: PMC2992502  PMID: 21067601
23.  Characterisation of phenotypes based on severity of emphysema in chronic obstructive pulmonary disease 
Thorax  2007;62(11):932-937.
Background
Airflow limitation in chronic obstructive pulmonary disease (COPD) is caused by a mixture of small airway disease and emphysema, the relative contributions of which may vary among patients. Phenotypes of COPD classified purely based on severity of emphysema are not well defined and may be different from the classic phenotypes of “pink puffers” and “blue bloaters”.
Methods
To characterise clinical phenotypes based on severity of emphysema, 274 subjects with COPD were recruited, excluding those with physician‐diagnosed bronchial asthma. For all subjects a detailed interview of disease history and symptoms, quality of life (QOL) measurement, blood sampling, pulmonary function tests before and after inhalation of salbutamol (0.4 mg) and high‐resolution CT scanning were performed.
Results
Severity of emphysema visually evaluated varied widely even among subjects with the same stage of disease. No significant differences were noted among three groups of subjects classified by severity of emphysema in age, smoking history, chronic bronchitis symptoms, blood eosinophil count, serum IgE level or bronchodilator response. However, subjects with severe emphysema had significantly lower body mass index (BMI) and poorer QOL scores, evaluated using St George's Respiratory Questionnaire (SGRQ), than those with no/mild emphysema (mean (SD) BMI 21.2 (0.5) vs 23.5 (0.3) kg/m2, respectively; SGRQ total score 40 (3) vs 28 (2), respectively; p<0.001 for both). These characteristics held true even if subjects with the same degree of airflow limitation were chosen.
Conclusions
The severity of emphysema varies widely even in patients with the same stage of COPD, and chronic bronchitis symptoms are equally distributed irrespective of emphysema severity. Patients with the phenotype in which emphysema predominates have lower BMI and poorer health‐related QOL.
doi:10.1136/thx.2006.072777
PMCID: PMC2117136  PMID: 17573447
24.  Mutational Analysis of the Poly(ADP-Ribosyl)ation Sites of the Transcription Factor CTCF Provides an Insight into the Mechanism of Its Regulation by Poly(ADP-Ribosyl)ation ▿ †  
Molecular and Cellular Biology  2009;30(5):1199-1216.
Poly(ADP-ribosyl)ation of the conserved multifunctional transcription factor CTCF was previously identified as important to maintain CTCF insulator and chromatin barrier functions. However, the molecular mechanism of this regulation and also the necessity of this modification for other CTCF functions remain unknown. In this study, we identified potential sites of poly(ADP-ribosyl)ation within the N-terminal domain of CTCF and generated a mutant deficient in poly(ADP-ribosyl)ation. Using this CTCF mutant, we demonstrated the requirement of poly(ADP-ribosyl)ation for optimal CTCF function in transcriptional activation of the p19ARF promoter and inhibition of cell proliferation. By using a newly generated isogenic insulator reporter cell line, the CTCF insulator function at the mouse Igf2-H19 imprinting control region (ICR) was found to be compromised by the CTCF mutation. The association and simultaneous presence of PARP-1 and CTCF at the ICR, confirmed by single and serial chromatin immunoprecipitation assays, were found to be independent of CTCF poly(ADP-ribosyl)ation. These results suggest a model of CTCF regulation by poly(ADP-ribosyl)ation whereby CTCF and PARP-1 form functional complexes at sites along the DNA, producing a dynamic reversible modification of CTCF. By using bioinformatics tools, numerous sites of CTCF and PARP-1 colocalization were demonstrated, suggesting that such regulation of CTCF may take place at the genome level.
doi:10.1128/MCB.00827-09
PMCID: PMC2820893  PMID: 20038529
25.  Cultivated Grapevines Represent a Symptomless Reservoir for the Transmission of Hop Stunt Viroid to Hop Crops: 15 Years of Evolutionary Analysis 
PLoS ONE  2009;4(12):e8386.
Hop stunt was a mysterious disorder that first emerged in the 1940s in commercial hops in Japan. To investigate the origin of this disorder, we infected hops with natural Hop stunt viroid (HpSVd) isolates derived from four host species (hop, grapevine, plum and citrus), which except for hop represent possible sources of the ancestral viroid. These plants were maintained for 15 years, then analyzed the HpSVd variants present. Here we show that the variant originally found in cultivated grapevines gave rise to various combinations of mutations at positions 25, 26, 54, 193, and 281. However, upon prolonged infection, these variants underwent convergent evolution resulting in a limited number of adapted mutants. Some of them showed nucleotide sequences identical to those currently responsible for hop stunt epidemics in commercial hops in Japan, China, and the United States. Therefore, these results indicate that we have successfully reproduced the original process by which a natural HpSVd variant naturally introduced into cultivated hops was able to mutate into the HpSVd variants that are currently present in commercial hops. Furthermore, and importantly, we have identified cultivated grapevines as a symptomless reservoir in which HSVd can evolve and be transmitted to hop crops to cause epidemics.
doi:10.1371/journal.pone.0008386
PMCID: PMC2793511  PMID: 20041179

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