Photocontrol of the expression of genes encoding chlorophyll a/b binding proteins and small subunit of ribulose-1,5-bisphosphate carboxylase in etiolated seedlings of Lycopersicon esculentum (L. ) and Nicotiana tabacum (L. )

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wehmeyer, B.; Cashmore, A.R.; Schaefer, E.

Phytochrome and the blue ultraviolet-A photoreceptor control light-induced expression of genes encoding the chlorophyll a/b binding protein of photosystem II and photosystem I and the genes for the small subunit of the ribulose-1,5-bisphosphate carboxylase in etiolated seedlings of Lycopersicon esculentum (tomato) and Nicotiana tabacum (tobacco). A high irradiance response also controls the induction of these genes. Genes encoding photosystem II- and I-associated chlorophyll a/b binding proteins both exhibit a transient rapid increase in expression in response to light pulse or to continuous irradiation. In contrast, genes encoding the small subunit exhibit a continuous increase in expression in response to light.more » These distinct expression characteristics are shown to reflect differences at the level of transcription.« less

Different effects of enhanced and reduced expression of pub gene on the formation of embryoid bodies by cultured embryonic mouse stem cell.

PubMed

Novosadova, E V; Manuilova, E S; Arsen'eva, E L; Khaidarova, N V; Dolotov, O V; Inozemtseva, L S; Kozachenkov, K Yu; Tarantul, V Z; Grivennikov, I A

2005-07-01

The effects of pub gene on proliferation and initial stages of differentiation of embryonic mouse stem cells were studied in vitro. To this end we used enhanced expression of human pub gene (hpub) and suppression of expression of mouse endogenous pub gene with RNA-interference in embryonic stem cells. Proliferative activity of genetically modified polyclonal lines of the embryonic stem cells transfected with plasmids carrying expressing hpub gene or plasmids generating small interference RNA to this gene did not differ from that of the control cells. Inhibition of expression of endogenous pub gene in embryonic stem cells using small interference RNA 2-fold decreased the formation of embryoid bodies, at the same time additional expression of exogenous hpub gene almost 2-fold increased their number in comparison with the control. It was hypothesized that pub gene participates in early stages of differentiation of embryonic stem cells leading to the formation of embryoid bodies.

The magnitude and colour of noise in genetic negative feedback systems.

PubMed

Voliotis, Margaritis; Bowsher, Clive G

2012-08-01

The comparative ability of transcriptional and small RNA-mediated negative feedback to control fluctuations or 'noise' in gene expression remains unexplored. Both autoregulatory mechanisms usually suppress the average (mean) of the protein level and its variability across cells. The variance of the number of proteins per molecule of mean expression is also typically reduced compared with the unregulated system, but is almost never below the value of one. This relative variance often substantially exceeds a recently obtained, theoretical lower limit for biochemical feedback systems. Adding the transcriptional or small RNA-mediated control has different effects. Transcriptional autorepression robustly reduces both the relative variance and persistence (lifetime) of fluctuations. Both benefits combine to reduce noise in downstream gene expression. Autorepression via small RNA can achieve more extreme noise reduction and typically has less effect on the mean expression level. However, it is often more costly to implement and is more sensitive to rate parameters. Theoretical lower limits on the relative variance are known to decrease slowly as a measure of the cost per molecule of mean expression increases. However, the proportional increase in cost to achieve substantial noise suppression can be different away from the optimal frontier-for transcriptional autorepression, it is frequently negligible.

[Arf6, RalA and BIRC5 protein expression in non small cell lung cancer].

PubMed

Knizhnik, A V; Kovaleva, O B; Laktionov, K K; Mochal'nikova, V V; Komel'kov, A V; Chevkina, E M; Zborovskaia, I B

2011-01-01

Evaluation of tumor markers expression pattern which determines individual progression parameters is one of the major topics in molecular oncopathology research. This work presents research on expression analysis of several Ras-Ral associated signal transduction pathway proteins (Arf6, RalA and BIRC5) in accordance with clinical criteria in non small cell lung cancer patients. Using Western-blot analysis and RT-PCR Arf6, RalA and BIRC5 expression has been analyzed in parallel in 53 non small cell lung cancer samples of different origin. Arf6 protein expression was elevated in 55% non small cell lung cancer tumor samples in comparison with normal tissue. In the group of squamous cell lung cancer Arf6 expression elevation was observed more often. RalA protein expression was decreased in comparison to normal tissue samples in 64% of non small cell lung cancer regardless to morphological structure. Correlation between RalA protein expression decrease and absence of regional metastases was revealed for squamous cell lung cancer. BIRC5 protein expression in tumor samples versus corresponding normal tissue was 1.3 times more often elevated in the squamous cell lung cancer group (in 76% tumor samples). At the same time elevation of BIRC5 expression was fixed only in 63% of adenocarcinoma tumor samples. A statistically significant decrease (p = 0.0158) of RalA protein expression and increase (p = 0.0498) of Arf6 protein expression in comparison with normal tissue was found for T1-2N0M0 and T1-2N1-2M0 groups of squamous cell lung cancer correspondingly.

Enteroendocrine K and L cells in healthy and type 2 diabetic individuals.

PubMed

Jorsal, Tina; Rhee, Nicolai A; Pedersen, Jens; Wahlgren, Camilla D; Mortensen, Brynjulf; Jepsen, Sara L; Jelsing, Jacob; Dalbøge, Louise S; Vilmann, Peter; Hassan, Hazem; Hendel, Jakob W; Poulsen, Steen S; Holst, Jens J; Vilsbøll, Tina; Knop, Filip K

2018-02-01

Enteroendocrine K and L cells are pivotal in regulating appetite and glucose homeostasis. Knowledge of their distribution in humans is sparse and it is unknown whether alterations occur in type 2 diabetes. We aimed to evaluate the distribution of enteroendocrine K and L cells and relevant prohormone-processing enzymes (using immunohistochemical staining), and to evaluate the mRNA expression of the corresponding genes along the entire intestinal tract in individuals with type 2 diabetes and healthy participants. In this cross-sectional study, 12 individuals with type 2 diabetes and 12 age- and BMI-matched healthy individuals underwent upper and lower double-balloon enteroscopy with mucosal biopsy retrieval from approximately every 30 cm of the small intestine and from seven specific anatomical locations in the large intestine. Significantly different densities for cells positive for chromogranin A (CgA), glucagon-like peptide-1, glucose-dependent insulinotropic polypeptide, peptide YY, prohormone convertase (PC) 1/3 and PC2 were observed along the intestinal tract. The expression of CHGA did not vary along the intestinal tract, but the mRNA expression of GCG, GIP, PYY, PCSK1 and PCSK2 differed along the intestinal tract. Lower counts of CgA-positive and PC1/3-positive cells, respectively, were observed in the small intestine of individuals with type 2 diabetes compared with healthy participants. In individuals with type 2 diabetes compared with healthy participants, the expression of GCG and PYY was greater in the colon, while the expression of GIP and PCSK1 was greater in the small intestine and colon, and the expression of PCSK2 was greater in the small intestine. Our findings provide a detailed description of the distribution of enteroendocrine K and L cells and the expression of their products in the human intestinal tract and demonstrate significant differences between individuals with type 2 diabetes and healthy participants. NCT03044860.

Involvement of Antizyme Characterized from the Small Abalone Haliotis diversicolor in Gonadal Development.

PubMed

Li, Wei-Dong; Huang, Min; Lü, Wen-Gang; Chen, Xiao; Shen, Ming-Hui; Li, Xiang-Min; Wang, Rong-Xia; Ke, Cai-Huan

2015-01-01

The small abalone Haliotis diversicolor is an economically important mollusk that is widely cultivated in Southern China. Gonad precocity may affect the aquaculture of small abalone. Polyamines, which are small cationic molecules essential for cellular proliferation, may affect gonadal development. Ornithine decarboxylase (ODC) and antizyme (AZ) are essential elements of a feedback circuit that regulates cellular polyamines. This paper presents the molecular cloning and characterization of AZ from small abalone. Sequence analysis showed that the cDNA sequence of H. diversicolor AZ (HdiODCAZ) consisted of two overlapping open reading frames (ORFs) and conformed to the +1 frameshift property of the frame. Thin Layer chromatography (TLC) analysis suggested that the expressed protein encoded by +1 ORF2 was the functional AZ that targets ODC to 26S proteasome degradation. The result demonstrated that the expression level of AZ was higher than that of ODC in the ovary of small abalone. In addition, the expression profiles of ODC and AZ at the different development stages of the ovary indicated that these two genes might be involved in the gonadal development of small abalone.

Involvement of Antizyme Characterized from the Small Abalone Haliotis diversicolor in Gonadal Development

PubMed Central

Lü, Wen-Gang; Chen, Xiao; Shen, Ming-Hui; Li, Xiang-Min; Wang, Rong-Xia; Ke, Cai-Huan

2015-01-01

The small abalone Haliotis diversicolor is an economically important mollusk that is widely cultivated in Southern China. Gonad precocity may affect the aquaculture of small abalone. Polyamines, which are small cationic molecules essential for cellular proliferation, may affect gonadal development. Ornithine decarboxylase (ODC) and antizyme (AZ) are essential elements of a feedback circuit that regulates cellular polyamines. This paper presents the molecular cloning and characterization of AZ from small abalone. Sequence analysis showed that the cDNA sequence of H. diversicolor AZ (HdiODCAZ) consisted of two overlapping open reading frames (ORFs) and conformed to the +1 frameshift property of the frame. Thin Layer chromatography (TLC) analysis suggested that the expressed protein encoded by +1 ORF2 was the functional AZ that targets ODC to 26S proteasome degradation. The result demonstrated that the expression level of AZ was higher than that of ODC in the ovary of small abalone. In addition, the expression profiles of ODC and AZ at the different development stages of the ovary indicated that these two genes might be involved in the gonadal development of small abalone. PMID:26313647

Small and big Hodgkin-Reed-Sternberg cells of Hodgkin lymphoma cell lines L-428 and L-1236 lack consistent differences in gene expression profiles and are capable to reconstitute each other.

PubMed

Rengstl, Benjamin; Kim, Sooji; Döring, Claudia; Weiser, Christian; Bein, Julia; Bankov, Katrin; Herling, Marco; Newrzela, Sebastian; Hansmann, Martin-Leo; Hartmann, Sylvia

2017-01-01

The hallmark of classical Hodgkin lymphoma (cHL) is the presence of giant, mostly multinucleated Hodgkin-Reed-Sternberg (HRS) cells. Whereas it has recently been shown that giant HRS cells evolve from small Hodgkin cells by incomplete cytokinesis and re-fusion of tethered sister cells, it remains unsolved why this phenomenon particularly takes place in this lymphoma and what the differences between these cell types of variable sizes are. The aim of the present study was to characterize microdissected small and giant HRS cells by gene expression profiling and to assess differences of clonal growth behavior as well as susceptibility toward cytotoxic intervention between these different cell types to provide more insight into their distinct cellular potential. Applying stringent filter criteria, only two differentially expressed genes between small and giant HRS cells, SHFM1 and LDHB, were identified. With looser filter criteria, 13 genes were identified to be differentially overexpressed in small compared to giant HRS cells. These were mainly related to energy metabolism and protein synthesis, further suggesting that small Hodgkin cells resemble the proliferative compartment of cHL. SHFM1, which is known to be involved in the generation of giant cells, was downregulated in giant RS cells at the RNA level. However, reduced mRNA levels of SHFM1, LDHB and HSPA8 did not translate into decreased protein levels in giant HRS cells. In cell culture experiments it was observed that the fraction of small and big HRS cells was adjusted to the basic level several days after enrichment of these populations via cell sorting, indicating that small and big HRS cells can reconstitute the full spectrum of cells usually observed in the culture. However, assessment of clonal growth of HRS cells indicated a significantly reduced potential of big HRS cells to form single cell colonies. Taken together, our findings pinpoint to strong similarities but also some differences between small and big HRS cells.

Sex differences in pain-related behavior and expression of calcium/calmodulin-dependent protein kinase II in dorsal root ganglia of rats with diabetes type 1 and type 2.

PubMed

Ferhatovic, Lejla; Banozic, Adriana; Kostic, Sandra; Sapunar, Damir; Puljak, Livia

2013-06-01

Sex differences in pain-related behavior and expression of calcium/calmodulin dependent protein kinase II (CaMKII) in dorsal root ganglia were studied in rat models of Diabetes mellitus type 1 (DM1) and type 2 (DM2). DM1 was induced with 55mg/kg streptozotocin, and DM2 with a combination of high-fat diet and 35mg/kg of streptozotocin. Pain-related behavior was analyzed using thermal and mechanical stimuli. The expression of CaMKII was analyzed with immunofluorescence. Sexual dimorphism in glycemia, and expression of CaMKII was observed in the rat model of DM1, but not in DM2 animals. Increased expression of total CaMKII (tCaMKII) in small-diameter dorsal root ganglia neurons, which are associated with nociception, was found only in male DM1 rats. None of the animals showed increased expression of the phosphorylated alpha CaMKII isoform in small-diameter neurons. The expression of gamma and delta isoforms of CaMKII remained unchanged in all analyzed animal groups. Different patterns of glycemia and tCaMKII expression in male and female model of DM1 were not associated with sexual dimorphism in pain-related behavior. The present findings do not suggest sex-related differences in diabetic painful peripheral neuropathy in male and female diabetic rats. Copyright © 2012 Elsevier GmbH. All rights reserved.

Expression of the monocarboxylate transporter 1 (MCT1) in cells of the porcine intestine.

PubMed

Welter, Harald; Claus, Rolf

2008-06-01

Uptake of energy into cells and its allocation to individual cellular compartments by transporters are essential for tissue homeostasis. The present study gives an analysis of MCT1 expression and its cellular occurrence in the porcine intestine. Tissue portions from duodenum, jejunum, ileum, colon ascendens, colon transversum and colon descendens were collected and prepared for immunohistochemistry, Western blot and real time RT-PCR. A 169bp porcine MCT1 cDNA fragment was amplified and published. MCT1 mRNA expression in the large intestine was 20 fold higher compared to the small intestine. Western blot detected a single protein band of 41kDa at a much higher amount of MCT1 protein in the large intestine vs. the small intestine. MCT1 protein was detected in mitochondrial fractions of the large but not the small intestine. Immunohistochemistry in the small intestine showed that immune cells in the lamina propria and in the lymphoid follicles primarily expressed MCT1 while in the colon epithelial cells were the main source of MCT1. In summary, cellular expression of MCT1 differs between epithelial cells in the colon and small intestine. A possible role of MCT1 for uptake of butyrate into immune cells and the overall role of MCT1 for intestinal immune cell function remains elusive.

The magnitude and colour of noise in genetic negative feedback systems

PubMed Central

Voliotis, Margaritis; Bowsher, Clive G.

2012-01-01

The comparative ability of transcriptional and small RNA-mediated negative feedback to control fluctuations or ‘noise’ in gene expression remains unexplored. Both autoregulatory mechanisms usually suppress the average (mean) of the protein level and its variability across cells. The variance of the number of proteins per molecule of mean expression is also typically reduced compared with the unregulated system, but is almost never below the value of one. This relative variance often substantially exceeds a recently obtained, theoretical lower limit for biochemical feedback systems. Adding the transcriptional or small RNA-mediated control has different effects. Transcriptional autorepression robustly reduces both the relative variance and persistence (lifetime) of fluctuations. Both benefits combine to reduce noise in downstream gene expression. Autorepression via small RNA can achieve more extreme noise reduction and typically has less effect on the mean expression level. However, it is often more costly to implement and is more sensitive to rate parameters. Theoretical lower limits on the relative variance are known to decrease slowly as a measure of the cost per molecule of mean expression increases. However, the proportional increase in cost to achieve substantial noise suppression can be different away from the optimal frontier—for transcriptional autorepression, it is frequently negligible. PMID:22581772

Gene expression profiling in respond to TBT exposure in small abalone Haliotis diversicolor.

PubMed

Jia, Xiwei; Zou, Zhihua; Wang, Guodong; Wang, Shuhong; Wang, Yilei; Zhang, Ziping

2011-10-01

In this study, we investigated the gene expression profiling of small abalone, Haliotis diversicolor by tributyltin (TBT) exposure using a cDNA microarray containing 2473 unique transcripts. Totally, 107 up-regulated genes and 41 down-regulated genes were found. For further investigation of candidate genes from microarray data and EST analysis, quantitative real-time PCR was performed at 6 h, 24 h, 48 h, 96 h and 192 h TBT exposure. 26 genes were found to be significantly differentially expressed in different time course, 3 of them were unknown. Some gene homologues like cellulose, endo-beta-1,4-glucanase, ferritin subunit 1 and thiolester containing protein II CG7052-PB might be the good biomarker candidate for TBT monitor. The identification of stress response genes and their expression profiles will permit detailed investigation of the defense responses of small abalone genes. Published by Elsevier Ltd.

A Tale of Two RNAs during Viral Infection: How Viruses Antagonize mRNAs and Small Non-Coding RNAs in The Host Cell

PubMed Central

Herbert, Kristina M.; Nag, Anita

2016-01-01

Viral infection initiates an array of changes in host gene expression. Many viruses dampen host protein expression and attempt to evade the host anti-viral defense machinery. Host gene expression is suppressed at several stages of host messenger RNA (mRNA) formation including selective degradation of translationally competent messenger RNAs. Besides mRNAs, host cells also express a variety of noncoding RNAs, including small RNAs, that may also be subject to inhibition upon viral infection. In this review we focused on different ways viruses antagonize coding and noncoding RNAs in the host cell to its advantage. PMID:27271653

Soybean and Fish Oil Mixture With Different ω-6/ω-3 Polyunsaturated Fatty Acid Ratios Modulates Dextran Sulfate Sodium-Induced Changes in Small Intestinal Intraepithelial γδT-Lymphocyte Expression in Mice.

PubMed

Pai, Man-Hui; Liu, Jun-Jen; Hou, Yu-Chen; Yeh, Chiu-Li

2016-03-01

This study investigated the effect of different ω-6/ω-3 polyunsaturated fatty acid (PUFA) ratios on dextran sulfate sodium (DSS)-induced changes to small intestinal intraepithelial lymphocyte (IEL) γδT-cell expression. Mice were assigned to 3 control and 3 DSS-treated groups and were maintained on a low-fat semipurified diet. One of the control (S) groups and a DSS (DS) group were provided with soybean oil; the other 2 control (Hω-3 and Lω-3) groups and 2 other DSS (DHω-3 and DLω-3) groups were fed either a soybean and fish oil mixture with a ω-6/ω-3 ratio of 2:1 or 4:1. After feeding the respective diets for 2 weeks, the DSS groups were given distilled water containing 2% DSS, and the control groups were given distilled water for 5 days. All groups were further provided distilled water 5 days for recovery, and the small intestinal IEL γδT-cell subset was isolated for analysis. DSS treatment resulted in a lower small intestinal IEL γδT-cell percentage and higher messenger RNA (mRNA) expressions of Reg IIIγ, keratinocyte growth factor (KGF), and complement 5a receptor (C5aR) by IEL γδT cells. Fish oil administration enhanced the proportion of small intestinal IEL γδT cells. Compared with the DLω-3 group, the DHω-3 group had lower Reg IIIγ, KGF, and C5aR mRNA expressions and higher expression of peroxisome proliferator-activated receptor (PPAR)-γ gene by small intestinal IEL γδT cells. Fish oil diets with a ω-6/ω-3 PUFA ratio of 2:1 were more effective than those with a ratio of 4:1 in improving DSS-induced small intestinal injury, and activation of PPAR-γ in IEL γδT cells may be associated with resolution of small intestinal inflammation. © 2014 American Society for Parenteral and Enteral Nutrition.

The small regulatory RNA FasX enhances group A Streptococcus virulence and inhibits pilus expression via serotype-specific targets

PubMed Central

Danger, Jessica L.; Cao, Tram N.; Cao, Tran H.; Sarkar, Poulomee; Treviño, Jeanette; Pflughoeft, Kathryn J.; Sumby, Paul

2015-01-01

Summary Bacterial pathogens commonly show intra-species variation in virulence factor expression and often this correlates with pathogenic potential. The group A Streptococcus (GAS) produces a small regulatory RNA (sRNA), FasX, which regulates the expression of pili and the thrombolytic agent streptokinase. As GAS serotypes are polymorphic regarding (a) FasX abundance, (b) the fibronectin, collagen, T-antigen (FCT) region of the genome, which contains the pilus genes (nine different FCT-types), and (c) the streptokinase-encoding gene (ska) sequence (two different alleles), we sought to test whether FasX regulates pilus and streptokinase expression in a serotype-specific manner. Parental, fasX mutant, and complemented derivatives of serotype M1 (ska-2, FCT-2), M2 (ska-1, FCT-6), M6 (ska-2, FCT-1), and M28 (ska-1, FCT-4) isolates were compared. While FasX reduced pilus expression in each serotype, the molecular basis differed, as FasX bound, and inhibited the translation of, different FCT-region mRNAs. FasX enhanced streptokinase expression in each serotype, although the degree of regulation varied. Finally, we established that the regulation afforded by FasX enhances GAS virulence, assessed by a model of bacteremia using human plasminogen-expressing mice. Our data are the first to identify and characterize serotype-specific regulation by an sRNA in GAS, and to show an sRNA directly contributes to GAS virulence. PMID:25586884

Microarray analysis to identify the similarities and differences of pathogenesis between aortic occlusive disease and abdominal aortic aneurysm.

PubMed

Wang, Guofu; Bi, Lechang; Wang, Gaofeng; Huang, Feilai; Lu, Mingjing; Zhu, Kai

2018-06-01

Objectives Expression profile of GSE57691 was analyzed to identify the similarities and differences between aortic occlusive disease and abdominal aortic aneurysm. Methods The expression profile of GSE57691 was downloaded from Gene Expression Omnibus database, including 20 small abdominal aortic aneurysm samples, 29 large abdominal aortic aneurysm samples, 9 aortic occlusive disease samples, and 10 control samples. Using the limma package in R, the differentially expressed genes were screened. Followed by enrichment analysis was performed for the differentially expressed genes using database for annotation, visualization, and integrated discovery online tool. Based on string online tool and Cytoscape software, protein-protein interaction network and module analyses were carried out. Moreover, integrated TF platform database and Cytoscape software were used for constructing transcriptional regulatory networks. Results As a result, 1757, 354, and 396 differentially expressed genes separately were identified in aortic occlusive disease, large abdominal aortic aneurysm, and small abdominal aortic aneurysm samples. UBB was significantly enriched in proteolysis related pathways with a high degree in three groups. SPARCL1 was another gene shared by these groups and regulated by NFIA, which had a high degree in transcriptional regulatory network. ACTB, a significant upregulated gene in abdominal aortic aneurysm samples, could be regulated by CLIC4, which was significantly enriched in cell motions. ACLY and NFIB were separately identified in aortic occlusive disease and small abdominal aortic aneurysm samples, and separately enriched in lipid metabolism and negative regulation of cell proliferation. Conclusions The downregulated UBB, NFIA, and SPARCL1 might play key roles in both aortic occlusive disease and abdominal aortic aneurysm, while the upregulated ACTB might only involve in abdominal aortic aneurysm. ACLY and NFIB were specifically involved in aortic occlusive disease and small abdominal aortic aneurysm separately.

Role of miRNAs and siRNAs in biotic and abiotic stress responses of plants

PubMed Central

Khraiwesh, Basel; Zhu, Jian-Kang; Zhu, Jianhua

2011-01-01

Small, non-coding RNAs are a distinct class of regulatory RNAs in plants and animals that control a variety of biological processes. In plants, several classes of small RNAs with specific sizes and dedicated functions have evolved through a series of pathways. The major classes of small RNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs), which differ in their biogenesis. miRNAs control the expression of cognate target genes by binding to reverse complementary sequences, resulting in cleavage or translational inhibition of the target RNAs. siRNAs have a similar structure, function, and biogenesis as miRNAs but are derived from long double-stranded RNAs and can often direct DNA methylation at target sequences. Besides their roles in growth and development and maintenance of genome integrity, small RNAs are also important components in plant stress responses. One way in which plants respond to environmental stress is by modifying their gene expression through the activity of small RNAs. Thus, understanding how small RNAs regulate gene expression will enable researchers to explore the role of small RNAs in biotic and abiotic stress responses. This review focuses on the regulatory roles of plant small RNAs in the adaptive response to stresses. PMID:21605713

The 18 kDa Translocator Protein (Peripheral Benzodiazepine Receptor) Expression in the Bone of Normal, Osteoprotegerin or Low Calcium Diet Treated Mice

PubMed Central

Kam, Winnie Wai-Ying; Meikle, Steven R.; Dunstan, Colin R.; Banati, Richard B.

2012-01-01

The presence of the translocator protein (TSPO), previously named as the mitochondrial or peripheral benzodiazepine receptor, in bone cells was studied in vitro and in situ using RT-qPCR, and receptor autoradiography using the selective TSPO ligand PK11195. In vitro, the TSPO is highly expressed in osteoblastic and osteoclastic cells. In situ, constitutive expression of TSPO is found in bone marrow and trabecular bone, e.g., spongiosa. Mice with a reduction of bone turnover induced by a 4-day treatment of osteoprotegerin reduces [3H]PK11195 binding in the spongiosa (320±128 Bq.mg−1, 499±106 Bq.mg−1 in saline-treated controls). In contrast, mice with an increase in bone turnover caused by a 4-day low calcium diet increases [3H]PK11195 binding in the spongiosa (615±90 Bq.mg−1). Further, our study includes technical feasibility data on [18F]fluoride microPET imaging of rodent bone with altered turnover. Despite [18F]fluoride having high uptake, the in vivo signal differences were small. Using a phantom model, we describe the spillover effect and partial volume loss that affect the quantitative microPET imaging of the small bone structures in experimental mouse models. In summary, we demonstrate the expression of TSPO in small rodent bone tissues, including osteoblasts and osteoclasts. A trend increase in TSPO expression was observed in the spongiosa from low to high bone turnover conditions. However, despite the potential utility of TSPO expression as an in vivo biomarker of bone turnover in experimental rodent models, our small animal PET imaging data using [18F]fluoride show that even under the condition of a good biological signal-to-noise ratio and high tracer uptake, the currently achievable instrument sensitivity and spatial resolution is unlikely to be sufficient to detect subtle differences in small structures, such as mouse bone. PMID:22295097

Differential Sensitivity of Target Genes to Translational Repression by miR-17~92

PubMed Central

Jin, Hyun Yong; Oda, Hiroyo; Chen, Pengda; Kang, Seung Goo; Valentine, Elizabeth; Liao, Lujian; Zhang, Yaoyang; Gonzalez-Martin, Alicia; Shepherd, Jovan; Head, Steven R.; Kim, Pyeung-Hyeun; Fu, Guo; Liu, Wen-Hsien; Han, Jiahuai

2017-01-01

MicroRNAs (miRNAs) are thought to exert their functions by modulating the expression of hundreds of target genes and each to a small degree, but it remains unclear how small changes in hundreds of target genes are translated into the specific function of a miRNA. Here, we conducted an integrated analysis of transcriptome and translatome of primary B cells from mutant mice expressing miR-17~92 at three different levels to address this issue. We found that target genes exhibit differential sensitivity to miRNA suppression and that only a small fraction of target genes are actually suppressed by a given concentration of miRNA under physiological conditions. Transgenic expression and deletion of the same miRNA gene regulate largely distinct sets of target genes. miR-17~92 controls target gene expression mainly through translational repression and 5’UTR plays an important role in regulating target gene sensitivity to miRNA suppression. These findings provide molecular insights into a model in which miRNAs exert their specific functions through a small number of key target genes. PMID:28241004

Single-cell RNA-sequencing reveals a distinct population of proglucagon-expressing cells specific to the mouse upper small intestine.

PubMed

Glass, Leslie L; Calero-Nieto, Fernando J; Jawaid, Wajid; Larraufie, Pierre; Kay, Richard G; Göttgens, Berthold; Reimann, Frank; Gribble, Fiona M

2017-10-01

To identify sub-populations of intestinal preproglucagon-expressing (PPG) cells producing Glucagon-like Peptide-1, and their associated expression profiles of sensory receptors, thereby enabling the discovery of therapeutic strategies that target these cell populations for the treatment of diabetes and obesity. We performed single cell RNA sequencing of PPG-cells purified by flow cytometry from the upper small intestine of 3 GLU-Venus mice. Cells from 2 mice were sequenced at low depth, and from the third mouse at high depth. High quality sequencing data from 234 PPG-cells were used to identify clusters by tSNE analysis. qPCR was performed to compare the longitudinal and crypt/villus locations of cluster-specific genes. Immunofluorescence and mass spectrometry were used to confirm protein expression. PPG-cells formed 3 major clusters: a group with typical characteristics of classical L-cells, including high expression of Gcg and Pyy (comprising 51% of all PPG-cells); a cell type overlapping with Gip-expressing K-cells (14%); and a unique cluster expressing Tph1 and Pzp that was predominantly located in proximal small intestine villi and co-produced 5-HT (35%). Expression of G-protein coupled receptors differed between clusters, suggesting the cell types are differentially regulated and would be differentially targetable. Our findings support the emerging concept that many enteroendocrine cell populations are highly overlapping, with individual cells producing a range of peptides previously assigned to distinct cell types. Different receptor expression profiles across the clusters highlight potential drug targets to increase gut hormone secretion for the treatment of diabetes and obesity. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

Global assessment of small RNAs reveals a non-coding transcript involved in biofilm formation and attachment in Acinetobacter baumannii ATCC 17978

PubMed Central

Pérez, Astrid; Gómez, Manuel J.; Gayoso, Carmen; Vallejo, Juan A.; Ohneck, Emily J.; Valle, Jaione; Actis, Luis A.; Beceiro, Alejandro; Bou, Germán

2017-01-01

Many strains of Acinetobacter baumannii have been described as being able to form biofilm. Small non-coding RNAs (sRNAs) control gene expression in many regulatory circuits in bacteria. The aim of the present work was to provide a global description of the sRNAs produced both by planktonic and biofilm-associated (sessile) cells of A. baumannii ATCC 17978, and to compare the corresponding gene expression profiles to identify sRNAs molecules associated to biofilm formation and virulence. sRNA was extracted from both planktonic and sessile cells and reverse transcribed. cDNA was subjected to 454-pyrosequencing using the GS-FLX Titanium chemistry. The global analysis of the small RNA transcriptome revealed different sRNA expression patterns in planktonic and biofilm associated cells, with some of the transcripts only expressed or repressed in sessile bacteria. A total of 255 sRNAs were detected, with 185 of them differentially expressed in the different types of cells. A total of 9 sRNAs were expressed only in biofilm cells, while the expression of other 21 coding regions were repressed only in biofilm cells. Strikingly, the expression level of the sRNA 13573 was 120 times higher in biofilms than in planktonic cells, an observation that prompted us to further investigate the biological role of this non-coding transcript. Analyses of an isogenic mutant and over-expressing strains revealed that the sRNA 13573 gene is involved in biofilm formation and attachment to A549 human alveolar epithelial cells. The present work serves as a basis for future studies examining the complex regulatory network that regulate biofilm biogenesis and attachment to eukaryotic cells in A. baumannii ATCC 17978. PMID:28763494

Potential miRNA regulators of differential HPG axis gene expression between low egg producing and high egg producing turkey hens

USDA-ARS?s Scientific Manuscript database

Expression differences exist in key genes of the hypothalamo-pituitary-gonadal (HPG) axis in low egg producing hens (LEPH) and high egg producing hens (HEPH); however, regulation of these differences is unknown. MicroRNAs (miRNAs) are small non-coding RNAs that play a role in post-transcriptional re...

The expression of COX-2, hTERT, MDM2, LATS2 and S100A2 in different types of non-small cell lung cancer (NSCLC).

PubMed

Strazisar, Mojca; Mlakar, Vid; Glavac, Damjan

2009-01-01

Several studies have reported different expression levels of certain genes in NSCLC, mostly related to the stage and advancement of the tumours. We investigated 65 stage I-III NSCLC tumours: 32 adenocarcinomas (ADC), 26 squamous cell carcinomas (SCC) and 7 large cell carcinomas (LCC). Using the real-time reverse transcription polymerase chain reaction (RT-PCR), we analysed the expression of the COX-2, hTERT, MDM2, LATS2 and S100A2 genes and researched the relationships between the NSCLC types and the differences in expression levels. The differences in the expression levels of the LATS2, S100A2 and hTERT genes in different types of NSCLC are significant. hTERT and COX-2 were over-expressed and LATS2 under-expressed in all NSCLC. We also detected significant relative differences in the expression of LATS2 and MDM2, hTERT and MDM2 in different types of NSCLC. There was a significant difference in the average expression levels in S100A2 for ADC and SCC. Our study shows differences in the expression patterns within the NSCLC group, which may mimic the expression of the individual NSCLC type, and also new relationships in the expression levels for different NSCLC types.

Multilevel Regulation of Bacterial Gene Expression with the Combined STAR and Antisense RNA System.

PubMed

Lee, Young Je; Kim, Soo-Jung; Moon, Tae Seok

2018-03-16

Synthetic small RNA regulators have emerged as a versatile tool to predictably control bacterial gene expression. Owing to their simple design principles, small size, and highly orthogonal behavior, these engineered genetic parts have been incorporated into genetic circuits. However, efforts to achieve more sophisticated cellular functions using RNA regulators have been hindered by our limited ability to integrate different RNA regulators into complex circuits. Here, we present a combined RNA regulatory system in Escherichia coli that uses small transcription activating RNA (STAR) and antisense RNA (asRNA) to activate or deactivate target gene expression in a programmable manner. Specifically, we demonstrated that the activated target output by the STAR system can be deactivated by expressing two different types of asRNAs: one binds to and sequesters the STAR regulator, affecting the transcription process, while the other binds to the target mRNA, affecting the translation process. We improved deactivation efficiencies (up to 96%) by optimizing each type of asRNA and then integrating the two optimized asRNAs into a single circuit. Furthermore, we demonstrated that the combined STAR and asRNA system can control gene expression in a reversible way and can regulate expression of a gene in the genome. Lastly, we constructed and simultaneously tested two A AND NOT B logic gates in the same cell to show sophisticated multigene regulation by the combined system. Our approach establishes a methodology for integrating multiple RNA regulators to rationally control multiple genes.

Molecular cloning and characterization of ADP-glucose pyrophosphorylase cDNA clones isolated from pea cotyledons.

PubMed

Burgess, D; Penton, A; Dunsmuir, P; Dooner, H

1997-02-01

Three ADP-glucose pyrophosphorylase (ADPG-PPase) cDNA clones have been isolated and characterized from a pea cotyledon cDNA library. Two of these clones (Psagps1 and Psagps2) encode the small subunit of ADPG-PPase. The deduced amino acid sequences for these two clones are 95% identical. Expression of these two genes differs in that the Psagps2 gene shows comparatively higher expression in seeds relative to its expression in other tissues. Psagps2 expression also peaks midway through seed development at a time in which Psagps1 transcripts are still accumulating. The third cDNA isolated (Psagp11) encodes the large subunit of ADPG-PPase. It shows greater selectivity in expression than either of the small subunit clones. It is highly expressed in sink organs (seed, pod, and seed coat) and undetectable in leaves.

High Expression of UGT1A1/1A6 in Monkey Small Intestine: Comparison of Protein Expression Levels of Cytochromes P450, UDP-Glucuronosyltransferases, and Transporters in Small Intestine of Cynomolgus Monkey and Human.

PubMed

Akazawa, Takanori; Uchida, Yasuo; Miyauchi, Eisuke; Tachikawa, Masanori; Ohtsuki, Sumio; Terasaki, Tetsuya

2018-01-02

Cynomolgus monkeys have been widely used for the prediction of drug absorption in humans. The purpose of this study was to clarify the regional protein expression levels of cytochromes P450 (CYPs), UDP-glucuronosyltransferases (UGTs), and transporters in small intestine of cynomolgus monkey using liquid chromatography-tandem mass spectrometry, and to compare them with the corresponding levels in human. UGT1A1 in jejunum and ileum were >4.57- and >3.11-fold and UGT1A6 in jejunum and ileum were >16.1- and >8.57-fold, respectively, more highly expressed in monkey than in human. Also, jejunal expression of monkey CYP3A8 (homologue of human CYP3A4) was >3.34-fold higher than that of human CYP3A4. Among apical drug efflux transporters, BCRP showed the most abundant expression in monkey and human, and the expression levels of BCRP in monkey and human were >1.74- and >1.25-fold greater than those of P-gp and >2.76- and >4.50-fold greater than those of MRP2, respectively. These findings should be helpful to understand species differences of the functions of CYPs, UGTs, and transporters between monkey and human. The UGT1A1/1A6 data would be especially important because it is difficult to identify isoforms responsible for species differences of intestinal glucuronidation by means of functional studies due to overlapping substrate specificity.

A Novel Class of Somatic Small RNAs Similar to Germ Cell Pachytene PIWI-interacting Small RNAs*

PubMed Central

Ortogero, Nicole; Schuster, Andrew S.; Oliver, Daniel K.; Riordan, Connor R.; Hong, Annie S.; Hennig, Grant W.; Luong, Dickson; Bao, Jianqiang; Bhetwal, Bhupal P.; Ro, Seungil; McCarrey, John R.; Yan, Wei

2014-01-01

PIWI-interacting RNAs (piRNAs) are small noncoding RNAs that bind PIWI family proteins exclusively expressed in the germ cells of mammalian gonads. MIWI2-associated piRNAs are essential for silencing transposons during primordial germ cell development, and MIWI-bound piRNAs are required for normal spermatogenesis during adulthood in mice. Although piRNAs have long been regarded as germ cell-specific, increasing lines of evidence suggest that somatic cells also express piRNA-like RNAs (pilRNAs). Here, we report the detection of abundant pilRNAs in somatic cells, which are similar to MIWI-associated piRNAs mainly expressed in pachytene spermatocytes and round spermatids in the testis. Based on small RNA deep sequencing and quantitative PCR analyses, pilRNA expression is dynamic and displays tissue specificity. Although pilRNAs are similar to pachytene piRNAs in both size and genomic origins, they have a distinct ping-pong signature. Furthermore, pilRNA biogenesis appears to utilize a yet to be identified pathway, which is different from all currently known small RNA biogenetic pathways. In addition, pilRNAs appear to preferentially target the 3′-UTRs of mRNAs in a partially complementary manner. Our data suggest that pilRNAs, as an integral component of the small RNA transcriptome in somatic cell lineages, represent a distinct population of small RNAs that may have functions similar to germ cell piRNAs. PMID:25320077

Selective accumulation of langerhans-type dendritic cells in small airways of patients with COPD

PubMed Central

2010-01-01

Background Dendritic cells (DC) linking innate and adaptive immune responses are present in human lungs, but the characterization of different subsets and their role in COPD pathogenesis remain to be elucidated. The aim of this study is to characterize and quantify pulmonary myeloid DC subsets in small airways of current and ex-smokers with or without COPD. Methods Myeloid DC were characterized using flowcytometry on single cell suspensions of digested human lung tissue. Immunohistochemical staining for langerin, BDCA-1, CD1a and DC-SIGN was performed on surgical resection specimens from 85 patients. Expression of factors inducing Langerhans-type DC (LDC) differentiation was evaluated by RT-PCR on total lung RNA. Results Two segregated subsets of tissue resident pulmonary myeloid DC were identified in single cell suspensions by flowcytometry: the langerin+ LDC and the DC-SIGN+ interstitial-type DC (intDC). LDC partially expressed the markers CD1a and BDCA-1, which are also present on their known blood precursors. In contrast, intDC did not express langerin, CD1a or BDCA-1, but were more closely related to monocytes. Quantification of DC in the small airways by immunohistochemistry revealed a higher number of LDC in current smokers without COPD and in COPD patients compared to never smokers and ex-smokers without COPD. Importantly, there was no difference in the number of LDC between current and ex-smoking COPD patients. In contrast, the number of intDC did not differ between study groups. Interestingly, the number of BDCA-1+ DC was significantly lower in COPD patients compared to never smokers and further decreased with the severity of the disease. In addition, the accumulation of LDC in the small airways significantly correlated with the expression of the LDC inducing differentiation factor activin-A. Conclusions Myeloid DC differentiation is altered in small airways of current smokers and COPD patients resulting in a selective accumulation of the LDC subset which correlates with the pulmonary expression of the LDC-inducing differentiation factor activin-A. This study identified the LDC subset as an interesting focus for future research in COPD pathogenesis. PMID:20307269

A Conserved Developmental Patterning Network Produces Quantitatively Different Output in Multiple Species of Drosophila

PubMed Central

Meyer, Miriah; Wunderlich, Zeba; Simirenko, Lisa; Luengo Hendriks, Cris L.; Keränen, Soile V. E.; Henriquez, Clara; Knowles, David W.; Biggin, Mark D.; Eisen, Michael B.; DePace, Angela H.

2011-01-01

Differences in the level, timing, or location of gene expression can contribute to alternative phenotypes at the molecular and organismal level. Understanding the origins of expression differences is complicated by the fact that organismal morphology and gene regulatory networks could potentially vary even between closely related species. To assess the scope of such changes, we used high-resolution imaging methods to measure mRNA expression in blastoderm embryos of Drosophila yakuba and Drosophila pseudoobscura and assembled these data into cellular resolution atlases, where expression levels for 13 genes in the segmentation network are averaged into species-specific, cellular resolution morphological frameworks. We demonstrate that the blastoderm embryos of these species differ in their morphology in terms of size, shape, and number of nuclei. We present an approach to compare cellular gene expression patterns between species, while accounting for varying embryo morphology, and apply it to our data and an equivalent dataset for Drosophila melanogaster. Our analysis reveals that all individual genes differ quantitatively in their spatio-temporal expression patterns between these species, primarily in terms of their relative position and dynamics. Despite many small quantitative differences, cellular gene expression profiles for the whole set of genes examined are largely similar. This suggests that cell types at this stage of development are conserved, though they can differ in their relative position by up to 3–4 cell widths and in their relative proportion between species by as much as 5-fold. Quantitative differences in the dynamics and relative level of a subset of genes between corresponding cell types may reflect altered regulatory functions between species. Our results emphasize that transcriptional networks can diverge over short evolutionary timescales and that even small changes can lead to distinct output in terms of the placement and number of equivalent cells. PMID:22046143

Tunable diblock copolypeptide hydrogel depots for local delivery of hydrophobic molecules in healthy and injured central nervous system

PubMed Central

Zhang, Shanshan; Anderson, Mark A.; Ao, Yan; Khakh, Baljit S.; Fan, Jessica; Deming, Timothy J.; Sofroniew, Michael V.

2014-01-01

Many hydrophobic small molecules are available to regulate gene expression and other cellular functions. Locally restricted application of such molecules in the central nervous system (CNS) would be desirable in many experimental and therapeutic settings, but is limited by a lack of innocuous vehicles able to load and easily deliver hydrophobic cargo. Here, we tested the potential for diblock copolypeptide hydrogels (DCH) to serve as such vehicles. In vitro tests on loading and release were conducted with cholesterol and the anti-cancer agent, temozolomide (TMZ). Loading of hydrophobic cargo modified DCH physical properties such as stiffness and viscosity, but these could readily be tuned to desired ranges by modifying DCH concentration, amino acid composition or chain lengths. Different DCH formulations exhibited different loading capacities and different rates of release. For example, comparison of different DCH with increasing alanine contents showed corresponding increases in both cargo loading capacity and time for cargo release. In vivo tests were conducted with tamoxifen, a small synthetic hydrophobic molecule widely used to regulate transgene expression. Tamoxifen released from DCH depots injected into healthy or injured CNS efficiently activated reporter gene expression in a locally restricted manner in transgenic mice. These findings demonstrate the facile and predictable tunability of DCH to achieve a wide range of loading capacities and release profiles of hydrophobic cargos while retaining CNS compatible physical properties. In addition, the findings show that DCH depots injected into the CNS can efficiently deliver small hydrophobic molecules that regulate gene expression in local cells. PMID:24314556

Novel Regulatory Small RNAs in Streptococcus pyogenes

PubMed Central

Tesorero, Rafael A.; Yu, Ning; Wright, Jordan O.; Svencionis, Juan P.; Cheng, Qiang; Kim, Jeong-Ho; Cho, Kyu Hong

2013-01-01

Streptococcus pyogenes (Group A Streptococcus or GAS) is a Gram-positive bacterial pathogen that has shown complex modes of regulation of its virulence factors to cause diverse diseases. Bacterial small RNAs are regarded as novel widespread regulators of gene expression in response to environmental signals. Recent studies have revealed that several small RNAs (sRNAs) have an important role in S. pyogenes physiology and pathogenesis by regulating gene expression at the translational level. To search for new sRNAs in S. pyogenes, we performed a genomewide analysis through computational prediction followed by experimental verification. To overcome the limitation of low accuracy in computational prediction, we employed a combination of three different computational algorithms (sRNAPredict, eQRNA and RNAz). A total of 45 candidates were chosen based on the computational analysis, and their transcription was analyzed by reverse-transcriptase PCR and Northern blot. Through this process, we discovered 7 putative novel trans-acting sRNAs. Their abundance varied between different growth phases, suggesting that their expression is influenced by environmental or internal signals. Further, to screen target mRNAs of an sRNA, we employed differential RNA sequencing analysis. This study provides a significant resource for future study of small RNAs and their roles in physiology and pathogenesis of S. pyogenes. PMID:23762235

Expression of K19 and K7 in dysplastic nodules and hepatocellular carcinoma.

PubMed

Bae, Jun Sang; Choi, Ha Na; Noh, Sang Jae; Park, Byung Hyun; Jang, Kyu Yun; Park, Cheol Keun; Moon, Woo Sung

2012-08-01

Hepatocellular carcinoma (HCC) is one of the most common types of malignant tumors characterized by a multistep process of tumor development. Nodular lesions that differ from the surrounding liver parenchyma and are characterized by cytological or structural atypia are termed dysplastic nodules (DNs). DNs are well-known precancerous HCC lesions. Expression of keratin (K) 19 and K7, molecular markers of hepatic progenitor cells and cholangiocytes, has been reported in certain HCCs. However, it remains unclear whether K19-positive HCC cells are derived from true hepatic progenitor cells or mature cells that have undergone a dedifferentiation or a transdifferentiation process. In total, 107 tissue sections (13 low-grade DNs, 15 high-grade DNs, 27 small HCCs and 52 large HCCs) from resected liver samples and 132 HCC tissue microarray (TMA) cores were subjected to immunohistochemical analysis for K19 and K7. Clinicopathological data of the HCC patients were evaluated. K19 expression was found in 0% of DNs, 19% of small HCCs (≤2 cm), 8% of large HCCs (>2 cm) and 8% of TMA samples. K7 expression was found in 14% of DNs, 41% of small HCCs, 15% of large HCCs and 6% of TMA samples. Among the five K19-positive small HCCs, four were distinctly nodular and one tumor was an infiltrative type. No vaguely nodular HCC was positive for K19. K19 expression was significantly associated with histological grade (P=0.023), serum α-fetoprotein level (P=0.001) and K7 expression (P=0.001) in HCC. K19 expression was an independent prognostic factor for overall survival in non-viral HCC patients (P=0.003). K19 expression is extremely rare in DNs and occurs in progressed small HCCs. Our results suggest that K19 expression may be an acquired feature of carcinoma cells during HCC progression in certain HCCs.

Differential gene expression in human abdominal aortic aneurysm and aortic occlusive disease

PubMed Central

Moran, Corey S.; Schreurs, Charlotte; Lindeman, Jan H. N.; Walker, Philip J.; Nataatmadja, Maria; West, Malcolm; Holdt, Lesca M.; Hinterseher, Irene; Pilarsky, Christian; Golledge, Jonathan

2015-01-01

Abdominal aortic aneurysm (AAA) and aortic occlusive disease (AOD) represent common causes of morbidity and mortality in elderly populations which were previously believed to have common aetiologies. The aim of this study was to assess the gene expression in human AAA and AOD. We performed microarrays using aortic specimen obtained from 20 patients with small AAAs (≤ 55mm), 29 patients with large AAAs (> 55mm), 9 AOD patients, and 10 control aortic specimens obtained from organ donors. Some differentially expressed genes were validated by quantitative-PCR (qRT-PCR)/immunohistochemistry. We identified 840 and 1,014 differentially expressed genes in small and large AAAs, respectively. Immune-related pathways including cytokine-cytokine receptor interaction and T-cell-receptor signalling were upregulated in both small and large AAAs. Examples of validated genes included CTLA4 (2.01-fold upregulated in small AAA, P = 0.002), NKTR (2.37-and 2.66-fold upregulated in small and large AAA with P = 0.041 and P = 0.015, respectively), and CD8A (2.57-fold upregulated in large AAA, P = 0.004). 1,765 differentially expressed genes were identified in AOD. Pathways upregulated in AOD included metabolic and oxidative phosphorylation categories. The UCP2 gene was downregulated in AOD (3.73-fold downregulated, validated P = 0.017). In conclusion, the AAA and AOD transcriptomes were very different suggesting that AAA and AOD have distinct pathogenic mechanisms. PMID:25944698

Distribution of binding sites for the plant lectin Ulex europaeus agglutinin I on primary sensory neurones in seven different mammalian species.

PubMed

Gerke, Michelle B; Plenderleith, Mark B

2002-01-01

There is an increasing body of evidence to suggest that different functional classes of neurones express characteristic cell-surface carbohydrates. Previous studies have shown that the plant lectin Ulex europaeus agglutinin-I (UEA) binds to a population of small to medium diameter primary sensory neurones in rabbits and humans. This suggests that a fucose-containing glycoconjugate may be expressed by nociceptive primary sensory neurones. In order to determine the extent to which this glycoconjugate is expressed by other species, in the current study, we have examined the distribution of UEA-binding sites on primary sensory neurones in seven different mammals. Binding sites for UEA were associated with the plasma membrane and cytoplasmic granules of small to medium dorsal root ganglion cells and their axon terminals in laminae I-III of the grey matter of the spinal cord, in the rabbit, cat and marmoset monkey. However, no binding was observed in either the dorsal root ganglia or spinal cord in the mouse, rat, guinea pig or flying fox. These results indicate an inter-species variation in the expression of cell-surface glycoconjugates on mammalian primary sensory neurones.

Identification of small non-coding RNA classes expressed in swine whole blood during HP-PRRSV infection.

PubMed

Fleming, Damarius S; Miller, Laura C

2018-04-01

It has been established that reduced susceptibility to porcine reproductive and respiratory syndrome virus (PRRSV) has a genetic component. This genetic component may take the form of small non-coding RNAs (sncRNA), which are molecules that function as regulators of gene expression. Various sncRNAs have emerged as having an important role in the immune system in humans. The study uses transcriptomic read counts to profile the type and quantity of both well and lesser characterized sncRNAs, such as microRNAs and small nucleolar RNAs to identify and quantify the classes of sncRNA expressed in whole blood between healthy and highly pathogenic PRRSV-infected pigs. Our results returned evidence on nine classes of sncRNA, four of which were consistently statistically significantly different based on Fisher's Exact Test, that can be detected and possibly interrogated for their effect on host dysregulation during PRRSV infections. Published by Elsevier Inc.

Identification of Small Molecules Targeting the Posttranscriptional Control of ERG Expression

DTIC Science & Technology

2012-10-01

ied. To establish a cell line expressing lucife rase-ERG fusion protein, the vector along pRL-CMV-Rluc expressing Renilla luciferase gene was...expanded, and examined for the e xpression of t wo different luciferases. A clone expressing both Firefly luciferase and Renilla luciferase was selected...treated with the individual chemical at 10 μM for 24 h. The dual luciferase activities were measured. The ratio of Firefly to Renilla lu ciferase

Differential replication dynamics for large and small Vibrio chromosomes affect gene dosage, expression and location

PubMed Central

Dryselius, Rikard; Izutsu, Kaori; Honda, Takeshi; Iida, Tetsuya

2008-01-01

Background Replication of bacterial chromosomes increases copy numbers of genes located near origins of replication relative to genes located near termini. Such differential gene dosage depends on replication rate, doubling time and chromosome size. Although little explored, differential gene dosage may influence both gene expression and location. For vibrios, a diverse family of fast growing gammaproteobacteria, gene dosage may be particularly important as they harbor two chromosomes of different size. Results Here we examined replication dynamics and gene dosage effects for the separate chromosomes of three Vibrio species. We also investigated locations for specific gene types within the genome. The results showed consistently larger gene dosage differences for the large chromosome which also initiated replication long before the small. Accordingly, large chromosome gene expression levels were generally higher and showed an influence from gene dosage. This was reflected by a higher abundance of growth essential and growth contributing genes of which many locate near the origin of replication. In contrast, small chromosome gene expression levels were low and appeared independent of gene dosage. Also, species specific genes are highly abundant and an over-representation of genes involved in transcription could explain its gene dosage independent expression. Conclusion Here we establish a link between replication dynamics and differential gene dosage on one hand and gene expression levels and the location of specific gene types on the other. For vibrios, this relationship appears connected to a polarisation of genetic content between its chromosomes, which may both contribute to and be enhanced by an improved adaptive capacity. PMID:19032792

The influence of cyclooxygenase-1 expression on the efficacy of cyclooxygenase-2 inhibition in head and neck squamous cell carcinoma cell lines.

PubMed

Park, Seok-Woo; Kim, Hyo-Sun; Choi, Myung-Sun; Kim, Ji-Eun; Jeong, Woo-Jin; Heo, Dae-Seog; Sung, Myung-Whun

2011-06-01

We have previously observed that cyclooxygenase-2 (COX-2) inhibition blocked the production of vascular endothelial growth factor (VEGF) in some head and neck squamous cell carcinoma (HNSCC) cells. However, as some HNSCC cells showed little response to COX-2 inhibition, although they highly expressed COX-2 and prostaglandin E2, we set out to elucidate what made this difference between them and focused on the possibility of the differential expression of COX-1. In western blotting, we found that COX-1 was expressed in SNU-1041 and SNU-1066, but not in SNU-1076 and PCI-50. Only in those cell lines without expression of COX-1 was VEGF production blocked meaningfully by small interfering RNA of COX-2. However, by cotreating with small interfering RNAs of COX-2 and COX-1, VEGF synthesis and prostaglandin E2 were inhibited in SNU-1041 and SNU-1066, similarly in SNU-1076 and PCI-50 with high expression of only COX-2. We also found that there was no difference in the pattern of prostaglandin synthesis between COX-2 and COX-1 through enzyme-linked immunosorbent assay for various prostaglandins. Our study suggests that, as COX-1 and COX-2 express and affect VEGF synthesis in HNSCC cells, we should check COX-1 expression in investigations on cancer treatment by inhibiting COX-2-induced prostaglandins.

Spatial influence on breast muscle morphological structure, myofiber size, and gene expression associated with the wooden breast myopathy in broilers.

PubMed

Clark, D L; Velleman, S G

2016-12-01

The wooden breast (WB) myopathy is identified by the palpation of a rigid pectoralis major (p. major) muscle and is characterized as a fibrotic, necrotic p. major disorder in broilers. The objective of the current study was to determine spatial morphological and gene expression differences at 4 locations within WB affected muscle from different genetic lines. Morphology was evaluated in 2 broiler lines expressing the WB myopathy (Lines A and B) and a line without WB (Line C) at 3 ventral locations and one anterodorsal location in the p. major muscle. In WB affected muscle of Line A, fibrosis was greatest in the anterior locations of WB affected muscle. In Line B muscle, fibrosis was greatest in the anteroventral region and minimal in the anterodorsal or posterior regions. Average p. major myofiber diameter was 30% larger in Lines A and B compared to Line C. However, in Line A there were no differences between the percentage of large fibers (diameter >70 μm) in unaffected and WB affected muscles at any sampling region. The percentage of small fibers (diameter <10 μm), likely small regenerating fibers, and expression of myogenic determination factor 1 (MYOD1) and myogenin were increased in Line A WB affected muscle compared to unaffected muscle. In Line B, the percentage of small fibers and MYOD1 expression in WB affected muscle was not different from unaffected muscle. Connective tissue organization within WB affected muscle was also different in Lines A and B, which may be attributed to decorin, a proteoglycan that mediates collagen crosslinking, growth factor signaling, and cell growth. Decorin expression was increased at all locations within Line A. However, in Line B decorin was increased only in the fibrotic regions of the p. major. The compiled results provide evidence that the WB myopathy is not uniform throughout the entire p. major muscle and the anterior end of the p. major muscle was more affected by the condition. © 2016 Poultry Science Association Inc.

Identification and Characterization of MicroRNAs in Ovary and Testis of Nile Tilapia (Oreochromis niloticus) by Using Solexa Sequencing Technology

PubMed Central

Zhou, Yi; Yu, Fan; Gao, Yun; Luo, Yongju; Tang, Zhanyang; Guo, Zhongbao; Guo, Enyan; Gan, Xi; Zhang, Ming; Zhang, Yaping

2014-01-01

MicroRNAs (miRNAs) are endogenous non-coding small RNAs which play important roles in the regulation of gene expression by cleaving or inhibiting the translation of target gene transcripts. Thereinto, some specific miRNAs show regulatory activities in gonad development via translational control. In order to further understand the role of miRNA-mediated posttranscriptional regulation in Nile tilapia (Oreochromis niloticus) ovary and testis, two small RNA libraries of Nile tilapia were sequenced by Solexa small RNA deep sequencing methods. A total of 9,731,431 and 8,880,497 raw reads, representing 5,407,800 and 4,396,281 unique sequences were obtained from the sexually mature ovaries and testes, respectively. After comparing the small RNA sequences with the Rfam database, 1,432,210 reads in ovaries and 984,146 reads in testes were matched to the genome sequence of Nile tilapia. Bioinformatic analysis identified 764 mature miRNA, 209 miRNA-5p and 202 miRNA-3p were found in the two libraries, of which 525 known miRNAs are both expressed in the ovary and testis of Nile tilapia. Comparison of expression profiles of the testis, miR-727, miR-129 and miR-29 families were highly expressed in tilapia ovary. Additionally, miR-132, miR-212, miR-33a and miR-135b families, showed significant higher expression in testis compared with that in ovary. Furthermore, the expression patterns of the miRNAs were analyzed in different developmental stages of gonad. The result showed different expression patterns were observed during development of testis and ovary. In addition, the identification and characterization of differentially expressed miRNAs in the ovaries and testis of Nile tilapia provides important information on the role of miRNA in the regulation of the ovarian and testicular development and function. This data will be helpful to facilitate studies on the regulation of miRNAs during teleosts reproduction. PMID:24466258

Staphylococcus aureus Regulatory RNAs as Potential Biomarkers for Bloodstream Infections

PubMed Central

Bordeau, Valérie; Cady, Anne; Revest, Matthieu; Rostan, Octavie; Sassi, Mohamed; Tattevin, Pierre; Donnio, Pierre-Yves

2016-01-01

Staphylococcus aureus is a commensal bacterium and pathogen. Identifying biomarkers for the transition from colonization to disease caused by this organism would be useful. Several S. aureus small RNAs (sRNAs) regulate virulence. We investigated presence and expression of 8 sRNAs in 83 S. aureus strains from 42 patients with sepsis or septic shock and 41 asymptomatic colonized carriers. Small pathogenicity island sRNAs sprB and sprC were clade specific. Six sRNAs had variable expression not correlated with clinical status. Expression of RNAIII was lower in strains from septic shock patients than in strains from colonized patients. When RNAIII was associated with expression of sprD, colonizing strains could be discriminated from strains in patients with bloodstream infections, including patients with sepsis and septic shock. Isolates associated with colonization might have sRNAs with target expression different from those of disease isolates. Monitoring expression of RNAIII and sprD could help determine severity of bloodstream infections. PMID:27224202

Staphylococcus aureus Regulatory RNAs as Potential Biomarkers for Bloodstream Infections.

PubMed

Bordeau, Valérie; Cady, Anne; Revest, Matthieu; Rostan, Octavie; Sassi, Mohamed; Tattevin, Pierre; Donnio, Pierre-Yves; Felden, Brice

2016-09-01

Staphylococcus aureus is a commensal bacterium and pathogen. Identifying biomarkers for the transition from colonization to disease caused by this organism would be useful. Several S. aureus small RNAs (sRNAs) regulate virulence. We investigated presence and expression of 8 sRNAs in 83 S. aureus strains from 42 patients with sepsis or septic shock and 41 asymptomatic colonized carriers. Small pathogenicity island sRNAs sprB and sprC were clade specific. Six sRNAs had variable expression not correlated with clinical status. Expression of RNAIII was lower in strains from septic shock patients than in strains from colonized patients. When RNAIII was associated with expression of sprD, colonizing strains could be discriminated from strains in patients with bloodstream infections, including patients with sepsis and septic shock. Isolates associated with colonization might have sRNAs with target expression different from those of disease isolates. Monitoring expression of RNAIII and sprD could help determine severity of bloodstream infections.

Progressive changes in non-coding RNA profile in leucocytes with age

PubMed Central

Muñoz-Culla, Maider; Irizar, Haritz; Gorostidi, Ana; Alberro, Ainhoa; Osorio-Querejeta, Iñaki; Ruiz-Martínez, Javier; Olascoaga, Javier; de Munain, Adolfo López; Otaegui, David

2017-01-01

It has been observed that immune cell deterioration occurs in the elderly, as well as a chronic low-grade inflammation called inflammaging. These cellular changes must be driven by numerous changes in gene expression and in fact, both protein-coding and non-coding RNA expression alterations have been observed in peripheral blood mononuclear cells from elder people. In the present work we have studied the expression of small non-coding RNA (microRNA and small nucleolar RNA -snoRNA-) from healthy individuals from 24 to 79 years old. We have observed that the expression of 69 non-coding RNAs (56 microRNAs and 13 snoRNAs) changes progressively with chronological age. According to our results, the age range from 47 to 54 is critical given that it is the period when the expression trend (increasing or decreasing) of age-related small non-coding RNAs is more pronounced. Furthermore, age-related miRNAs regulate genes that are involved in immune, cell cycle and cancer-related processes, which had already been associated to human aging. Therefore, human aging could be studied as a result of progressive molecular changes, and different age ranges should be analysed to cover the whole aging process. PMID:28448962

Engineered Saccharomyces cerevisiae strain for improved xylose utilization with a three-plasmid SUMO yeast expression system

USDA-ARS?s Scientific Manuscript database

A three-plasmid yeast expression system utilizing the portable small ubiquitin-like modifier (SUMO) vector set combined with the efficient endogenous yeast protease Ulp1 was developed for production of large amounts of soluble functional protein in Saccharomyces cerevisiae. Each vector has a differ...

Sex-Biased Gene Expression and Sexual Conflict throughout Development

PubMed Central

Ingleby, Fiona C.; Flis, Ilona; Morrow, Edward H.

2015-01-01

Sex-biased gene expression is likely to account for most sexually dimorphic traits because males and females share much of their genome. When fitness optima differ between sexes for a shared trait, sexual dimorphism can allow each sex to express their optimum trait phenotype, and in this way, the evolution of sex-biased gene expression is one mechanism that could help to resolve intralocus sexual conflict. Genome-wide patterns of sex-biased gene expression have been identified in a number of studies, which we review here. However, very little is known about how sex-biased gene expression relates to sex-specific fitness and about how sex-biased gene expression and conflict vary throughout development or across different genotypes, populations, and environments. We discuss the importance of these neglected areas of research and use data from a small-scale experiment on sex-specific expression of genes throughout development to highlight potentially interesting avenues for future research. PMID:25376837

Gene selection and cancer type classification of diffuse large-B-cell lymphoma using a bivariate mixture model for two-species data.

PubMed

Su, Yuhua; Nielsen, Dahlia; Zhu, Lei; Richards, Kristy; Suter, Steven; Breen, Matthew; Motsinger-Reif, Alison; Osborne, Jason

2013-01-05

: A bivariate mixture model utilizing information across two species was proposed to solve the fundamental problem of identifying differentially expressed genes in microarray experiments. The model utility was illustrated using a dog and human lymphoma data set prepared by a group of scientists in the College of Veterinary Medicine at North Carolina State University. A small number of genes were identified as being differentially expressed in both species and the human genes in this cluster serve as a good predictor for classifying diffuse large-B-cell lymphoma (DLBCL) patients into two subgroups, the germinal center B-cell-like diffuse large B-cell lymphoma and the activated B-cell-like diffuse large B-cell lymphoma. The number of human genes that were observed to be significantly differentially expressed (21) from the two-species analysis was very small compared to the number of human genes (190) identified with only one-species analysis (human data). The genes may be clinically relevant/important, as this small set achieved low misclassification rates of DLBCL subtypes. Additionally, the two subgroups defined by this cluster of human genes had significantly different survival functions, indicating that the stratification based on gene-expression profiling using the proposed mixture model provided improved insight into the clinical differences between the two cancer subtypes.

[Impact of high-fat diet induced obesity on glucose absorption in small intestinal mucose in rats].

PubMed

Huang, Wei; Liu, Rui; Guo, Wei; Wei, Na; Qiang, Ou; Li, Xian; Ou, Yan; Tang, Chengwei

2012-11-01

To investigate whether high-fat diet induced obesity was associated with variation of glucose absorption in small intestinal mucosa of rats. 46 male SD rats were randomly divided into high-fat diet group (n = 31) and control group (n = 15), fed with high-fat diet and normal diet for 24 weeks, respectively. After 24 weeks, the rats were divided into obese (n = 16) and obesity-resistant group (n = 10) according to their body weight. Rats' body weight, abdominal fat weight, plasma glucose level, maltase, sucrase activity in small intestinal mucosa were measured. SGLT-1 expression in intestinal mucosa was detected by immunohistochemistry, RT-PCR and Western blot. Mean body weight, abdominal fat weight, fast plasma glucose levels, maltase activities and SGLT-1 protein expression in intestinal mucosa of obese rats were significantly higher than those in the control and obesity-resistant rats (P < 0.05). Sucrase activities in intestinal mucosa showed no statistical difference among the three groups (P > 0.05). The SGLT-1 mRNA expression in obese group was increased by 12.5% and 23% when compare with the control and obesity-resistant group, respectively. But the difference was not statistical significant (P > 0.05). High-fat diet induced obesity was associated with the increased intestinal maltase activity and expression of SGLT-1 in rats, the key molecule in glucose absorption.

Isolation of Small SSEA-4-Positive Putative Stem Cells from the Ovarian Surface Epithelium of Adult Human Ovaries by Two Different Methods

PubMed Central

Virant-Klun, Irma; Skutella, Thomas; Hren, Matjaz; Gruden, Kristina; Cvjeticanin, Branko; Vogler, Andrej; Sinkovec, Jasna

2013-01-01

The adult ovarian surface epithelium has already been proposed as a source of stem cells and germinal cells in the literature, therefore it has been termed the “germinal epithelium”. At present more studies have confirmed the presence of stem cells expressing markers of pluripotency in adult mammalian ovaries, including humans. The aim of this study was to isolate a population of stem cells, based on the expression of pluripotency-related stage-specific embryonic antigen-4 (SSEA-4) from adult human ovarian surface epithelium by two different methods: magnetic-activated cell sorting and fluorescence-activated cell sorting. Both methods made it possible to isolate a similar, relatively homogenous population of small, SSEA-4-positive cells with diameters of up to 4 μm from the suspension of cells retrieved by brushing of the ovarian cortex biopsies in reproductive-age and postmenopausal women and in women with premature ovarian failure. The immunocytochemistry and genetic analyses revealed that these small cells—putative stem cells—expressed some primordial germ cell and pluripotency-related markers and might be related to the in vitro development of oocyte-like cells expressing some oocyte-specific transcription factors in the presence of donated follicular fluid with substances important for oocyte growth and development. The stemness of these cells needs to be further researched. PMID:23509763

The anti-apoptotic BAG3 protein is expressed in lung carcinomas and regulates small cell lung carcinoma (SCLC) tumor growth.

PubMed

Chiappetta, Gennaro; Basile, Anna; Barbieri, Antonio; Falco, Antonia; Rosati, Alessandra; Festa, Michelina; Pasquinelli, Rosa; Califano, Daniela; Palma, Giuseppe; Costanzo, Raffaele; Barcaroli, Daniela; Capunzo, Mario; Franco, Renato; Rocco, Gaetano; Pascale, Maria; Turco, Maria Caterina; De Laurenzi, Vincenzo; Arra, Claudio

2014-08-30

BAG3, member the HSP70 co-chaperones family, has been shown to play a relevant role in the survival, growth and invasiveness of different tumor types. In this study, we investigate the expression of BAG3 in 66 specimens from different lung tumors and the role of this protein in small cell lung cancer (SCLC) tumor growth. Normal lung tissue did not express BAG3 while we detected the expression of BAG3 by immunohistochemistry in all the 13 squamous cell carcinomas, 13 adenocarcinomas and 4 large cell carcinomas. Furthermore, we detected BAG3 expression in 22 of the 36 SCLCs analyzed. The role on SCLC cell survival was determined by down-regulating BAG3 levels in two human SCLC cell lines, i.e. H69 and H446, in vitro and measuring cisplatin induced apoptosis. Indeed down-regulation of BAG3 determines increased cell death and sensitizes cells to cisplatin treatment. The effect of BAG3 down-regulation on tumor growth was also investigated in an in vivo xenograft model by treating mice with an adenovirus expressing a specific bag3 siRNA. Treatment with bag3 siRNA-Ad significantly reduced tumor growth and improved animal survival. In conclusion we show that a subset of SCLCs over express BAG3 that exerts an anti-apoptotic effect resulting in resistance to chemotherapy.

Identification and verification of potential piRNAs from domesticated yak testis.

PubMed

Gong, Jishang; Zhang, Quanwei; Wang, Qi; Ma, Youji; Du, Jiaxiang; Zhang, Yong; Zhao, Xingxu

2018-02-01

PIWI-interacting RNAs (piRNA) are small non-coding RNA molecules expressed in animal germ cells that interact with PIWI family proteins to form RNA-protein complexes involved in epigenetic and post-transcriptional gene silencing of retrotransposons and other genetic elements in germ line cells, including reproductive stem cell self-sustainment, differentiation, meiosis and spermatogenesis. In the present study, we performed high-throughput sequencing of piRNAs in testis samples from yaks in different stages of sexual maturity. Deep sequencing of the small RNAs (18-40 nt in length) yielded 4,900,538 unique reads from a total of 53,035,635 reads. We identified yak small RNAs (18-30 nt) and performed functional characterization. Yak small RNAs showed a bimodal length distribution, with two peaks at 22 nt and >28 nt. More than 80% of the 3,106,033 putative piRNAs were mapped to 4637 piRNA-producing genomic clusters using RPKM. 6388 candidate piRNAs were identified from clean reads and the annotations were compared with the yak reference genome repeat region. Integrated network analysis suggested that some differentially expressed genes were involved in spermatogenesis through ECM-receptor interaction and PI3K-Akt signaling pathways. Our data provide novel insights into the molecular expression and regulation similarities and diversities in spermatogenesis and testicular development in yaks at different stages of sexual maturity. © 2018 The authors.

Identification and verification of potential piRNAs from domesticated yak testis

PubMed Central

Gong, Jishang; Zhang, Quanwei; Wang, Qi; Ma, Youji; Du, Jiaxiang; Zhang, Yong

2018-01-01

PIWI-interacting RNAs (piRNA) are small non-coding RNA molecules expressed in animal germ cells that interact with PIWI family proteins to form RNA–protein complexes involved in epigenetic and post-transcriptional gene silencing of retrotransposons and other genetic elements in germ line cells, including reproductive stem cell self-sustainment, differentiation, meiosis and spermatogenesis. In the present study, we performed high-throughput sequencing of piRNAs in testis samples from yaks in different stages of sexual maturity. Deep sequencing of the small RNAs (18–40 nt in length) yielded 4,900,538 unique reads from a total of 53,035,635 reads. We identified yak small RNAs (18–30 nt) and performed functional characterization. Yak small RNAs showed a bimodal length distribution, with two peaks at 22 nt and >28 nt. More than 80% of the 3,106,033 putative piRNAs were mapped to 4637 piRNA-producing genomic clusters using RPKM. 6388 candidate piRNAs were identified from clean reads and the annotations were compared with the yak reference genome repeat region. Integrated network analysis suggested that some differentially expressed genes were involved in spermatogenesis through ECM–receptor interaction and PI3K-Akt signaling pathways. Our data provide novel insights into the molecular expression and regulation similarities and diversities in spermatogenesis and testicular development in yaks at different stages of sexual maturity. PMID:29101267

Digestive enzyme expression and epithelial structure of small intestine in neonatal rats after 16 days spaceflight

NASA Astrophysics Data System (ADS)

Miyake, M.; Yamasaki, M.; Hazama, A.; Ijiri, K.; Shimizu, T.

It is important to assure whether digestive system can develop normally in neonates during spaceflight. Because the small intestine changes its function and structure drastically around weaning known as redifferentiation. Lactase expression declines and sucrase increases in small intestine for digestion of solid food before weaning. In this paper, we compared this enzyme transition and structural development of small intestine in neonatal rats after spaceflight. To find digestive genes differentially expressed in fight rats, DNA membrane macroarray was also used. Eight-day old rats were loaded to Space Shuttle Columbia, and housed in the animal facility for 16 days in space (STS-90, Neurolab mission). Two control groups (AGC; asynchronous ground control and VIV; vivarium) against flight group (FLT) were prepared. There was no difference in structure (crypt depth) and cell differentiation of epithelium between FLT and AGC by immunohistochemical analysis. We found that the amount of sucrase mRNA compared to lactase was decreased in FLT by RT-PCR. It reflected the enzyme transition was inhibited. Increase of 5 genes (APO A-I, APO A-IV, ACE, aFABP and aminopeptidase M) and decrease of carboxypeptidase-D were detected in FLT using macroarray. We think nutrition differences (less nourishment and late weaning) during spaceflight may cause inhibition of enzyme transition at least partly. The weightlessness might contribute to the inhibition through behavioral change.

Programmed death ligand 1 expression and CD8+ tumor-infiltrating lymphocyte density differences between paired primary and brain metastatic lesions in non-small cell lung cancer.

PubMed

Zhou, Jie; Gong, Zhihua; Jia, Qingzhu; Wu, Yan; Yang, Zhen-Zhou; Zhu, Bo

2018-04-15

Immunotherapy targeting the programmed cell death-1/programmed death ligand 1(PD-L1) pathway has shown promising antitumor activity in brain metastases (BMs) of non-small cell lung cancer (NSCLC) patients with an acceptable safety profile; however, the response rates often differ between primary lesions and intracranial lesions. Studies are necessary to identify detailed characterizations of the response biomarkers. In this study, we aimed to compare the differences of PD-L1 expression and CD8 + tumor-infiltrating lymphocyte (TIL) density, two major response biomarkers of PD-1/PD-L1 blockade, between paired primary and brain metastatic lesions in advanced NSCLC. We observed that among primary lesions or BMs, only a small number of patients harbored common PD-L1 expression on both tumor cells and tumor-infiltrating immune cells. Additionally, we found that the numbers of CD8 + TILs were significantly fewer in BMs than in primary lung cancers. Low stromal CD8 + TIL numbers in BMs were associated with significantly shorter overall survival compared to high stromal CD8 + TIL counts. Notably, we demonstrated a discrepancy in PD-L1 expression and CD8 + TIL density between primary lung cancers and their corresponding BMs. Such heterogeneities are significantly associated with the time at which BMs occurred. Our study emphasizes the spatial and temporal heterogeneity of biomarkers for anti-PD-1/PD-L1 therapy, which should be concerned in clinical practice. Copyright © 2018 Elsevier Inc. All rights reserved.

Intracellular Bioinorganic Chemistry and Cross Talk Among Different -Omics.

PubMed

Mendola, Diego La; Giacomelli, Chiara; Rizzarelli, Enrico

2016-01-01

The description of the cell life needs not only the knowledge of its genome and proteome, but also of the location of the metal ions and their different complex species in the subcellular compartments, that is of metallome. The cross-talk among these players of the omics' world secures the cellular homeostasis by means of a complex network, the alteration of which may give rise to many diseases. Copper and zinc ions levels regulate protein expression and metal-responsive transcription factors and in many pathologies metal dyshomeostasis induces to aberrant expression of different factors. microRNAs, a class of a small non-coding RNA molecules, act as RNA silencing and post-transcriptional regulators of gene expression contributing also to metal regulatory activity. The aim of the present review is to present how metals dyshomeostasis can be cause of diseases, involving different and specific metal chaperones, metal transporters, metalloproteins, small molecules and metal-sensing transcription factors. Two distinct classes of pathologies, cancer and osteoarthritis, are discussed starting from the metallostasis (metal homeostasis) and turning up to miRNAs regulation. The understanding of post-translational regulation, driven by metal ions sensing, may help to identify more specific targets and drugs to pathologies in which metal ions are involved.

High-Throughput Platform for Patient-Derived, Small Cell Number, Three-Dimensional Ovarian Cancer Spheroids

DTIC Science & Technology

2014-09-01

these small cell number spheroids show 3-D morphology (Figure 3). We also observed differences in the expression of mesenchymal markers when...Scale bar =100 µm. Figure 3: Small cell number spheroids demonstrate 3-D morphology . 3-D reconstructions of confocal z-stacks are shown for...formation was observed with the addition of MSCs, and subsequent co-culture in hanging drop plates preserved spheroid morphology indicated in the phase

FASTmiR: an RNA-based sensor for in vitro quantification and live-cell localization of small RNAs

PubMed Central

Huang, Kun; Doyle, Francis; Wurz, Zachary E.; Tenenbaum, Scott A.; Hammond, Reza K.

2017-01-01

Abstract Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), play a variety of important regulatory roles in many eukaryotes. Their small size has made it challenging to study them directly in live cells. Here we describe an RNA-based fluorescent sensor for small RNA detection both in vitro and in vivo, adaptable for any small RNA. It utilizes an sxRNA switch for detection of miRNA–mRNA interactions combined with a fluorophore-binding sequence ‘Spinach’, a GFP-like RNA aptamer for which the RNA–fluorophore complex exhibits strong and consistent fluorescence under an excitation wavelength. Two example sensors, FASTmiR171 and FASTmiR122, can rapidly detect and quantify the levels of miR171 and miR122 in vitro. The sensors can determine relative levels of miRNAs in total RNA extracts with sensitivity similar to small RNA sequencing and northern blots. FASTmiR sensors were also used to estimate the copy number range of miRNAs in total RNA extracts. To localize and analyze the spatial distribution of small RNAs in live, single cells, tandem copies of FASTmiR122 were expressed in different cell lines. FASTmiR122 was able to quantitatively detect the differences in miR122 levels in Huh7 and HEK293T cells demonstrating its potential for tracking miRNA expression and localization in vivo. PMID:28586459

Oocytes from small and large follicles exhibit similar development competence following goat cloning despite their differences in meiotic and cytoplasmic maturation.

PubMed

Yang, Min; Hall, Justin; Fan, Zhiqiang; Regouski, Misha; Meng, Qinggang; Rutigliano, Heloisa M; Stott, Rusty; Rood, Kerry A; Panter, Kip E; Polejaeva, Irina A

2016-12-01

Reduced developmental competence after IVF has been reported using oocyte derived from small follicles in several species including cattle, sheep, and goats. No information is currently available about the effect of follicle size of the cytoplast donor on in vivo development after somatic cell nuclear transfer (SCNT) in goats. Oocytes collected from large (≥3 mm) and small follicles (<3 mm) were examined for maturation and in vivo developmental competence after SCNT. Significantly greater maturation rate was observed in oocytes derived from large follicles compared with that of small follicles (51.6% and 33.7%, P < 0.05). Greater percent of large follicle oocytes exhibited a low glucose-6-phosphate dehydrogenase activity at germinal vesicle stage compared with small follicle oocytes (54.9% and 38.7%, P < 0.05). Relative mRNA expression analysis of 48 genes associated with embryonic and fetal development revealed that three genes (MATER, IGF2R, and GRB10) had higher level of expression in metaphase II oocytes from large follicles compared with oocytes from small follicles. Nevertheless, no difference was observed in pregnancy rates (33.3% vs. 47.1%) and birth rates (22.2% vs. 16.7%) after SCNT between the large and small follicle groups). These results indicate that metaphase II cytoplasts from small and large follicles have similar developmental competence when used in goat SCNT. Copyright © 2016 Elsevier Inc. All rights reserved.

Gene expression profiling of bovine ovarian follicular and luteal cells provides insight into cellular identities and functions

USDA-ARS?s Scientific Manuscript database

After ovulation, somatic cells of the ovarian follicle (theca and granulosa cells) become the small and large luteal cells of the corpus luteum. Aside from known cell type-specific receptors and steroidogenic enzymes, little is known about the differences in the gene expression profiles of these fou...

Influence of low protein diets on gene expression of digestive enzymes and hormone secretion in the gastrointestinal tract of young weaned piglets.

PubMed

Tian, Zhi-Mei; Ma, Xian-Yong; Yang, Xue-Fen; Fan, Qiu-Li; Xiong, Yun-Xia; Qiu, Yue-Qin; Wang, Li; Wen, Xiao-Lu; Jiang, Zong-Yong

To investigate dietary protein level effects on digestive mechanisms, weaned piglets were fed for 45 d with diets containing 20%, 17%, or 14% crude protein (CP) supplemented to meet requirements for essential amino acids. This article describes the influence of dietary protein on gastrointestinal hormones and expression of an array of digestive enzymes in the gastrointestinal tract and pancreas. Results indicated that there were no significant differences in expression of enzymes involved in carbohydrate digestion, except for maltase in the duodenum. In the jejunum, amylase expression in pigs fed 20% CP was much higher than that in pigs fed other diets (P<0.05) and maltase expression in those fed 17% CP was higher than that in other treatments (P<0.05). Although there were no remarkable differences in expression of aminopeptidase in the small intestine or carboxypeptidase in the pancreas (P>0.05), there was a trend towards higher expression of various proteases in pigs fed 17% CP. The duodenal expression of enteropeptidase in diets with 14% and 17% CP was significantly higher than that with 20% CP (P<0.05), but treatment differences did not existed in jejunum (P>0.05). The expression of GPR93 as a nutrient-responsive G protein-coupled receptor in 14% and 17% CP diets was significantly higher than that in 20% CP diet in the small intestine (P<0.05). The expressions of genes for pancreatic enzymes, lipase and elastase, were significantly higher in pigs fed diets with low CP, while similar trends occurred for carboxypeptidase, chymotrypsin and amylase. Conversely, the gastric expressions of pepsinogen A and progastricsin were lower with the 17% CP diet. Differences between treatments were found in the gastric antral contents of cholecystokinin and somatostatin: both increased in pigs fed 17% CP, accompanied by decreased content of motilin, which was also seen in plasma concentrations. These patterns were not reflected in duodenal contents. In general, 17% dietary CP was beneficial to the digestion of nutrient substance in the gastrointestinal tract.

Macromolecule exchange in Cuscuta-host plant interactions.

PubMed

Kim, Gunjune; Westwood, James H

2015-08-01

Cuscuta species (dodders) are parasitic plants that are able to grow on many different host plants and can be destructive to crops. The connections between Cuscuta and its hosts allow movement of not only water and small nutrients, but also macromolecules including mRNA, proteins and viruses. Recent studies show that RNAs move bidirectionally between hosts and parasites and involve a large number of different genes. Although the function of mobile mRNAs has not been demonstrated in this system, small RNAs are also transmitted and a silencing construct expressed in hosts is able to affect expression of the target gene in the parasite. High throughput sequencing of host-parasite associations has the potential to greatly accelerate understanding of this remarkable interaction. Copyright © 2015 Elsevier Ltd. All rights reserved.

Small RNA-based prediction of hybrid performance in maize.

PubMed

Seifert, Felix; Thiemann, Alexander; Schrag, Tobias A; Rybka, Dominika; Melchinger, Albrecht E; Frisch, Matthias; Scholten, Stefan

2018-05-21

Small RNA (sRNA) sequences are known to have a broad impact on gene regulation by various mechanisms. Their performance for the prediction of hybrid traits has not yet been analyzed. Our objective was to analyze the relation of parental sRNA expression with the performance of their hybrids, to develop a sRNA-based prediction approach, and to compare it to more common SNP and mRNA transcript based predictions using a factorial mating scheme of a maize hybrid breeding program. Correlation of genomic differences and messenger RNA (mRNA) or sRNA expression differences between parental lines with hybrid performance of their hybrids revealed that sRNAs showed an inverse relationship in contrast to the other two data types. We associated differences for SNPs, mRNA and sRNA expression between parental inbred lines with the performance of their hybrid combinations and developed two prediction approaches using distance measures based on associated markers. Cross-validations revealed parental differences in sRNA expression to be strong predictors for hybrid performance for grain yield in maize, comparable to genomic and mRNA data. The integration of both positively and negatively associated markers in the prediction approaches enhanced the prediction accurary. The associated sRNAs belong predominantly to the canonical size classes of 22- and 24-nt that show specific genomic mapping characteristics. Expression profiles of sRNA are a promising alternative to SNPs or mRNA expression profiles for hybrid prediction, especially for plant species without reference genome or transcriptome information. The characteristics of the sRNAs we identified suggest that association studies based on breeding populations facilitate the identification of sRNAs involved in hybrid performance.

Interspecific and host-related gene expression patterns in nematode-trapping fungi.

PubMed

Andersson, Karl-Magnus; Kumar, Dharmendra; Bentzer, Johan; Friman, Eva; Ahrén, Dag; Tunlid, Anders

2014-11-11

Nematode-trapping fungi are soil-living fungi that capture and kill nematodes using special hyphal structures called traps. They display a large diversity of trapping mechanisms and differ in their host preferences. To provide insights into the genetic basis for this variation, we compared the transcriptome expressed by three species of nematode-trapping fungi (Arthrobotrys oligospora, Monacrosporium cionopagum and Arthrobotrys dactyloides, which use adhesive nets, adhesive branches or constricting rings, respectively, to trap nematodes) during infection of two different plant-pathogenic nematode hosts (the root knot nematode Meloidogyne hapla and the sugar beet cyst nematode Heterodera schachtii). The divergence in gene expression between the fungi was significantly larger than that related to the nematode species being infected. Transcripts predicted to encode secreted proteins and proteins with unknown function (orphans) were overrepresented among the highly expressed transcripts in all fungi. Genes that were highly expressed in all fungi encoded endopeptidases, such as subtilisins and aspartic proteases; cell-surface proteins containing the carbohydrate-binding domain WSC; stress response proteins; membrane transporters; transcription factors; and transcripts containing the Ricin-B lectin domain. Differentially expressed transcripts among the fungal species encoded various lectins, such as the fungal fruit-body lectin and the D-mannose binding lectin; transcription factors; cell-signaling components; proteins containing a WSC domain; and proteins containing a DUF3129 domain. A small set of transcripts were differentially expressed in infections of different host nematodes, including peptidases, WSC domain proteins, tyrosinases, and small secreted proteins with unknown function. This is the first study on the variation of infection-related gene expression patterns in nematode-trapping fungi infecting different host species. A better understanding of these patterns will facilitate the improvements of these fungi in biological control programs, by providing molecular markers for screening programs and candidates for genetic manipulations of virulence and host preferences.

Differential expression of the eight genes of the petunia ribulose bisphosphate carboxylase small subunit multi-gene family

PubMed Central

Dean, Caroline; Elzen, Peter van den; Tamaki, Stanley; Dunsmuir, Pamela; Bedbrook, John

1985-01-01

Of the eight nuclear genes in the plant multi-gene family which encodes the small subunit (rbcS) of Petunia (Mitchell) ribulose bisphosphate carboxylase, one rbcS gene accounts for 47% of the total rbcS gene expression in petunia leaf tissue. Expression of each of five other rbcS genes is detected at levels between 2 and 23% of the total rbcS expression in leaf tissue, while expression of the remaining two rbcS genes is not detected. There is considerable variation (500-fold) in the levels of total rbcS mRNA in six organs of petunia (leaves, sepals, petals, stems, roots and stigmas/anthers). One gene, SSU301, showed the highest levels of steady-state mRNA in each of the organs examined. We discuss the differences in the steady-state mRNA levels of the individual rbcS genes in relation to their gene structure, nucleotide sequence and genomic linkage. ImagesFig. 2.Fig. 3. PMID:16453647

Novel population of small tumour-initiating stem cells in the ovaries of women with borderline ovarian cancer

PubMed Central

Virant-Klun, Irma; Stimpfel, Martin

2016-01-01

Small stem cells with diameters of up to 5 μm previously isolated from adult human ovaries indicated pluripotency and germinal lineage, especially primordial germ cells, and developed into primitive oocyte-like cells in vitro. Here, we show that a comparable population of small stem cells can be found in the ovarian tissue of women with borderline ovarian cancer, which, in contrast to small stem cells in “healthy” ovaries, formed spontaneous tumour-like structures and expressed some markers related to pluripotency and germinal lineage. The gene expression profile of these small putative cancer stem cells differed from similar cells sorted from “healthy” ovaries by 132 upregulated and 97 downregulated genes, including some important forkhead box and homeobox genes related to transcription regulation, developmental processes, embryogenesis, and ovarian cancer. These putative cancer stem cells are suggested to be a novel population of ovarian tumour-initiating cells in humans. PMID:27703207

Deep sequencing of small RNA libraries reveals dynamic expression patterns of microRNAs in multiple developmental stages of Bactrocera dorsalis.

PubMed

Huang, Y; Dou, W; Liu, B; Wei, D; Liao, C Y; Smagghe, G; Wang, J-J

2014-10-01

In eukaryotes, microRNAs (miRNAs) are small, conserved, noncoding RNAs that have emerged as critical regulators of gene expression. The oriental fruit fly Bactrocera dorsalis is one of the most economically important fruit fly pests in East Asia and the Pacific. Although transcriptome analyses have greatly enriched our knowledge of its structural genes, little is known about post-transcriptional regulation by miRNAs in this dipteran species. In this study, small RNA libraries corresponding to four B. dorsalis developmental stages (eggs, larvae, pupae and adults) were constructed and sequenced. Approximately 30.7 million reads of 18-30 nucleotides were obtained, with 123 known miRNAs and 60 novel miRNAs identified amongst these libraries. More than half of the miRNAs were stage-specific during the four developmental stages. A set of miRNAs was found to be up- or down-regulated during development by comparison of their reads at different developmental stages. Moreover, a small part of miRNAs owned both miR-#-3p and miR-#-5p types, with enormously variable miR-#-3p/miR-#-5p ratios in the same library and amongst different developmental stages for each miRNA. Taking these findings together, the current study has uncovered a number of miRNAs and provided insights into their possible involvement in developmental regulation by expression profiling of miRNAs. Further analyses of the expression and function of these miRNAs could increase our understanding of regulatory networks in this insect and lead to novel approaches for its control. © 2014 The Royal Entomological Society.

ALK-rearrangements and testing methods in non-small cell lung cancer: a review

PubMed Central

Shackelford, Rodney E.; Vora, Moiz; Mayhall, Kim; Cotelingam, James

2014-01-01

The anaplastic lymphoma tyrosine kinase (ALK) gene was first described as a driver mutation in anaplastic non-Hodgkin's lymphoma. Dysregulated ALK expression is now an identified driver mutation in nearly twenty different human malignancies, including 4-9% of non-small cell lung cancers (NSCLC). The tyrosine kinase inhibitor crizotinib is more effective than standard chemotherapeutic agents in treating ALK positive NSCLC, making molecular diagnostic testing for dysregulated ALK expression a necessary step in identifying optimal treatment modalities. Here we review ALKmediated signal transduction pathways and compare the molecular protocols used to identify dysregulated ALK expression in NSCLC. We also discuss the use of crizotinib and second generation ALK tyrosine kinase inhibitors in the treatment of ALK positive NSCLC, and the known mechanisms of crizotinib resistance in NSCLC. PMID:24955213

GmDREB1 overexpression affects the expression of microRNAs in GM wheat seeds

PubMed Central

Niu, Fengjuan; Hu, Zheng; Chen, Rui; Zhang, Hui

2017-01-01

MicroRNAs (miRNAs) are small regulators of gene expression that act on many different molecular and biochemical processes in eukaryotes. To date, miRNAs have not been considered in the current evaluation system for GM crops. In this study, small RNAs from the dry seeds of a GM wheat line overexpressing GmDREB1 and non-GM wheat cultivars were investigated using deep sequencing technology and bioinformatic approaches. As a result, 23 differentially expressed miRNAs in dry seeds were identified and confirmed between GM wheat and a non-GM acceptor. Notably, more differentially expressed tae-miRNAs between non-GM wheat varieties were found, indicating that the degree of variance between non-GM cultivars was considerably higher than that induced by the transgenic event. Most of the target genes of these differentially expressed miRNAs between GM wheat and a non-GM acceptor were associated with abiotic stress, in accordance with the product concept of GM wheat in improving drought and salt tolerance. Our data provided useful information and insights into the evaluation of miRNA expression in edible GM crops. PMID:28459812

GmDREB1 overexpression affects the expression of microRNAs in GM wheat seeds.

PubMed

Jiang, Qiyan; Sun, Xianjun; Niu, Fengjuan; Hu, Zheng; Chen, Rui; Zhang, Hui

2017-01-01

MicroRNAs (miRNAs) are small regulators of gene expression that act on many different molecular and biochemical processes in eukaryotes. To date, miRNAs have not been considered in the current evaluation system for GM crops. In this study, small RNAs from the dry seeds of a GM wheat line overexpressing GmDREB1 and non-GM wheat cultivars were investigated using deep sequencing technology and bioinformatic approaches. As a result, 23 differentially expressed miRNAs in dry seeds were identified and confirmed between GM wheat and a non-GM acceptor. Notably, more differentially expressed tae-miRNAs between non-GM wheat varieties were found, indicating that the degree of variance between non-GM cultivars was considerably higher than that induced by the transgenic event. Most of the target genes of these differentially expressed miRNAs between GM wheat and a non-GM acceptor were associated with abiotic stress, in accordance with the product concept of GM wheat in improving drought and salt tolerance. Our data provided useful information and insights into the evaluation of miRNA expression in edible GM crops.

Tissue distribution and effects of fasting and obesity on the ghrelin axis in mice.

PubMed

Morash, Michael G; Gagnon, Jeffrey; Nelson, Stephanie; Anini, Younes

2010-08-09

Ghrelin is a 28 amino acid peptide hormone derived from the 117 amino acid proghrelin, following cleavage by proprotein convertase 1 (PC1). In this study, we comprehensively assessed the tissue distribution and the effect of fasting and obesity on preproghrelin, Exon-4D, PC1 and GOAT expression and proghrelin-derived peptide (PGDP) secretion. The stomach was the major source of preproghrelin expression and PDGPs, followed by the small intestine. The remaining peripheral tissues (including the brain and pancreas) contained negligible expression levels. We detected obestatin in all stomach proghrelin cells, however, 22% of proghrelin cells in the small intestine did not express obestatin. There were strain differences in ghrelin secretion in response to fasting between CD1 and C57BL/6 mice. After a 24 hour-fast, CD1 mice had increased plasma levels of total ghrelin and obestatin with no change in preproghrelin mRNA or PGDP tissues levels. C57BL/6 mice showed a different response to a 24 hour-fast having increased proghrelin mRNA expression, stomach acylated ghrelin peptide and no change in plasma obestatin in C57BL/6 mice. In obese mice (ob/ob and diet-induced obesity (DIO)) there was a significant increase in preproghrelin mRNA levels while tissue and plasma PGDP levels were significantly reduced. Fasting did not affect PGDP in obese mice. Obese models displayed differences in GOAT expression, which was elevated in DIO mice, but reduced in ob/ob mice. We did not find co-localization of the leptin receptor in ghrelin expressing stomach cells, ruling out a direct effect of leptin on stomach ghrelin synthesis and secretion. Copyright (c) 2010 Elsevier B.V. All rights reserved.

A comparison of brain gene expression levels in domesticated and wild animals.

PubMed

Albert, Frank W; Somel, Mehmet; Carneiro, Miguel; Aximu-Petri, Ayinuer; Halbwax, Michel; Thalmann, Olaf; Blanco-Aguiar, Jose A; Plyusnina, Irina Z; Trut, Lyudmila; Villafuerte, Rafael; Ferrand, Nuno; Kaiser, Sylvia; Jensen, Per; Pääbo, Svante

2012-09-01

Domestication has led to similar changes in morphology and behavior in several animal species, raising the question whether similarities between different domestication events also exist at the molecular level. We used mRNA sequencing to analyze genome-wide gene expression patterns in brain frontal cortex in three pairs of domesticated and wild species (dogs and wolves, pigs and wild boars, and domesticated and wild rabbits). We compared the expression differences with those between domesticated guinea pigs and a distant wild relative (Cavia aperea) as well as between two lines of rats selected for tameness or aggression towards humans. There were few gene expression differences between domesticated and wild dogs, pigs, and rabbits (30-75 genes (less than 1%) of expressed genes were differentially expressed), while guinea pigs and C. aperea differed more strongly. Almost no overlap was found between the genes with differential expression in the different domestication events. In addition, joint analyses of all domesticated and wild samples provided only suggestive evidence for the existence of a small group of genes that changed their expression in a similar fashion in different domesticated species. The most extreme of these shared expression changes include up-regulation in domesticates of SOX6 and PROM1, two modulators of brain development. There was almost no overlap between gene expression in domesticated animals and the tame and aggressive rats. However, two of the genes with the strongest expression differences between the rats (DLL3 and DHDH) were located in a genomic region associated with tameness and aggression, suggesting a role in influencing tameness. In summary, the majority of brain gene expression changes in domesticated animals are specific to the given domestication event, suggesting that the causative variants of behavioral domestication traits may likewise be different.

A Comparison of Brain Gene Expression Levels in Domesticated and Wild Animals

PubMed Central

Albert, Frank W.; Somel, Mehmet; Carneiro, Miguel; Aximu-Petri, Ayinuer; Halbwax, Michel; Thalmann, Olaf; Blanco-Aguiar, Jose A.; Trut, Lyudmila; Villafuerte, Rafael; Ferrand, Nuno; Kaiser, Sylvia; Jensen, Per; Pääbo, Svante

2012-01-01

Domestication has led to similar changes in morphology and behavior in several animal species, raising the question whether similarities between different domestication events also exist at the molecular level. We used mRNA sequencing to analyze genome-wide gene expression patterns in brain frontal cortex in three pairs of domesticated and wild species (dogs and wolves, pigs and wild boars, and domesticated and wild rabbits). We compared the expression differences with those between domesticated guinea pigs and a distant wild relative (Cavia aperea) as well as between two lines of rats selected for tameness or aggression towards humans. There were few gene expression differences between domesticated and wild dogs, pigs, and rabbits (30–75 genes (less than 1%) of expressed genes were differentially expressed), while guinea pigs and C. aperea differed more strongly. Almost no overlap was found between the genes with differential expression in the different domestication events. In addition, joint analyses of all domesticated and wild samples provided only suggestive evidence for the existence of a small group of genes that changed their expression in a similar fashion in different domesticated species. The most extreme of these shared expression changes include up-regulation in domesticates of SOX6 and PROM1, two modulators of brain development. There was almost no overlap between gene expression in domesticated animals and the tame and aggressive rats. However, two of the genes with the strongest expression differences between the rats (DLL3 and DHDH) were located in a genomic region associated with tameness and aggression, suggesting a role in influencing tameness. In summary, the majority of brain gene expression changes in domesticated animals are specific to the given domestication event, suggesting that the causative variants of behavioral domestication traits may likewise be different. PMID:23028369

The Center for Optimized Structural Studies (COSS) platform for automation in cloning, expression, and purification of single proteins and protein-protein complexes.

PubMed

Mlynek, Georg; Lehner, Anita; Neuhold, Jana; Leeb, Sarah; Kostan, Julius; Charnagalov, Alexej; Stolt-Bergner, Peggy; Djinović-Carugo, Kristina; Pinotsis, Nikos

2014-06-01

Expression in Escherichia coli represents the simplest and most cost effective means for the production of recombinant proteins. This is a routine task in structural biology and biochemistry where milligrams of the target protein are required in high purity and monodispersity. To achieve these criteria, the user often needs to screen several constructs in different expression and purification conditions in parallel. We describe a pipeline, implemented in the Center for Optimized Structural Studies, that enables the systematic screening of expression and purification conditions for recombinant proteins and relies on a series of logical decisions. We first use bioinformatics tools to design a series of protein fragments, which we clone in parallel, and subsequently screen in small scale for optimal expression and purification conditions. Based on a scoring system that assesses soluble expression, we then select the top ranking targets for large-scale purification. In the establishment of our pipeline, emphasis was put on streamlining the processes such that it can be easily but not necessarily automatized. In a typical run of about 2 weeks, we are able to prepare and perform small-scale expression screens for 20-100 different constructs followed by large-scale purification of at least 4-6 proteins. The major advantage of our approach is its flexibility, which allows for easy adoption, either partially or entirely, by any average hypothesis driven laboratory in a manual or robot-assisted manner.

Oxidative stress gene expression profile in inbred mouse after ischemia/reperfusion small bowel injury.

PubMed

Bertoletto, Paulo Roberto; Ikejiri, Adauto Tsutomu; Somaio Neto, Frederico; Chaves, José Carlos; Teruya, Roberto; Bertoletto, Eduardo Rodrigues; Taha, Murched Omar; Fagundes, Djalma José

2012-11-01

To determine the profile of gene expressions associated with oxidative stress and thereby contribute to establish parameters about the role of enzyme clusters related to the ischemia/reperfusion intestinal injury. Twelve male inbred mice (C57BL/6) were randomly assigned: Control Group (CG) submitted to anesthesia, laparotomy and observed by 120 min; Ischemia/reperfusion Group (IRG) submitted to anesthesia, laparotomy, 60 min of small bowel ischemia and 60 min of reperfusion. A pool of six samples was submitted to the qPCR-RT protocol (six clusters) for mouse oxidative stress and antioxidant defense pathways. On the 84 genes investigated, 64 (76.2%) had statistic significant expression and 20 (23.8%) showed no statistical difference to the control group. From these 64 significantly expressed genes, 60 (93.7%) were up-regulated and 04 (6.3%) were down-regulated. From the group with no statistical significantly expression, 12 genes were up-regulated and 8 genes were down-regulated. Surprisingly, 37 (44.04%) showed a higher than threefold up-regulation and then arbitrarily the values was considered as a very significant. Thus, 37 genes (44.04%) were expressed very significantly up-regulated. The remained 47 (55.9%) genes were up-regulated less than three folds (35 genes - 41.6%) or down-regulated less than three folds (12 genes - 14.3%). The intestinal ischemia and reperfusion promote a global hyper-expression profile of six different clusters genes related to antioxidant defense and oxidative stress.

Changes in the Peripheral Blood Gene Expression Profile Induced by 3 Months of Valproate Treatment in Patients with Newly Diagnosed Epilepsy

PubMed Central

Rakitin, Aleksei; Kõks, Sulev; Reimann, Ene; Prans, Ele; Haldre, Sulev

2015-01-01

Valproic acid (VPA) is a widely used antiepileptic drug with a broad range of effects and broad clinical efficacy. As a well-known histone deacetylase (HDAC) inhibitor, VPA regulates epigenetic programming by altering the expression of many genes. The aim of study was to analyze differences in gene expression profiles before and after the start of VPA treatment in patients with newly diagnosed epilepsy. RNA sequencing was used to compare whole-genome gene expression patterns of peripheral blood from nine patients with epilepsy before and 3 months after the start of treatment with VPA. Of the 23,099 analyzed genes, only 11 showed statistically significant differential expression with false discovery rate-adjusted p-values below 0.1. Functional annotation and network analyses showed activation of only one genetic network (enrichment score = 30), which included genes for cardiovascular system development and function, cell morphology, and hematological system development and function. The finding of such a small number of differently expressed genes between before and after the start of treatment suggests a lack of HDAC inhibition in these patients, which could be explained by the relatively low doses of VPA that were used. In conclusion, VPA at standard therapeutic dosages modulates the expression of a small number of genes. Therefore, to minimize the potential side effects of HDAC inhibition, it is recommended that the lowest effective dose of VPA be used for treating epilepsy. PMID:26379622

tRF2Cancer: A web server to detect tRNA-derived small RNA fragments (tRFs) and their expression in multiple cancers.

PubMed

Zheng, Ling-Ling; Xu, Wei-Lin; Liu, Shun; Sun, Wen-Ju; Li, Jun-Hao; Wu, Jie; Yang, Jian-Hua; Qu, Liang-Hu

2016-07-08

tRNA-derived small RNA fragments (tRFs) are one class of small non-coding RNAs derived from transfer RNAs (tRNAs). tRFs play important roles in cellular processes and are involved in multiple cancers. High-throughput small RNA (sRNA) sequencing experiments can detect all the cellular expressed sRNAs, including tRFs. However, distinguishing genuine tRFs from RNA fragments generated by random degradation remains a major challenge. In this study, we developed an integrated web-based computing system, tRF2Cancer, to accurately identify tRFs from sRNA deep-sequencing data and evaluate their expression in multiple cancers. The binomial test was introduced to evaluate whether reads from a small RNA-seq data set represent tRFs or degraded fragments. A classification method was then used to annotate the types of tRFs based on their sites of origin in pre-tRNA or mature tRNA. We applied the pipeline to analyze 10 991 data sets from 32 types of cancers and identified thousands of expressed tRFs. A tool called 'tRFinCancer' was developed to facilitate the users to inspect the expression of tRFs across different types of cancers. Another tool called 'tRFBrowser' shows both the sites of origin and the distribution of chemical modification sites in tRFs on their source tRNA. The tRF2Cancer web server is available at http://rna.sysu.edu.cn/tRFfinder/. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Extent, Causes, and Consequences of Small RNA Expression Variation in Human Adipose Tissue

PubMed Central

Knights, Andrew J.; Abreu-Goodger, Cei; van de Bunt, Martijn; Guerra-Assunção, José Afonso; Bartonicek, Nenad; van Dongen, Stijn; Mägi, Reedik; Nisbet, James; Barrett, Amy; Rantalainen, Mattias; Nica, Alexandra C.; Quail, Michael A.; Small, Kerrin S.; Glass, Daniel; Enright, Anton J.; Winn, John; Deloukas, Panos; Dermitzakis, Emmanouil T.; McCarthy, Mark I.; Spector, Timothy D.; Durbin, Richard; Lindgren, Cecilia M.

2012-01-01

Small RNAs are functional molecules that modulate mRNA transcripts and have been implicated in the aetiology of several common diseases. However, little is known about the extent of their variability within the human population. Here, we characterise the extent, causes, and effects of naturally occurring variation in expression and sequence of small RNAs from adipose tissue in relation to genotype, gene expression, and metabolic traits in the MuTHER reference cohort. We profiled the expression of 15 to 30 base pair RNA molecules in subcutaneous adipose tissue from 131 individuals using high-throughput sequencing, and quantified levels of 591 microRNAs and small nucleolar RNAs. We identified three genetic variants and three RNA editing events. Highly expressed small RNAs are more conserved within mammals than average, as are those with highly variable expression. We identified 14 genetic loci significantly associated with nearby small RNA expression levels, seven of which also regulate an mRNA transcript level in the same region. In addition, these loci are enriched for variants significant in genome-wide association studies for body mass index. Contrary to expectation, we found no evidence for negative correlation between expression level of a microRNA and its target mRNAs. Trunk fat mass, body mass index, and fasting insulin were associated with more than twenty small RNA expression levels each, while fasting glucose had no significant associations. This study highlights the similar genetic complexity and shared genetic control of small RNA and mRNA transcripts, and gives a quantitative picture of small RNA expression variation in the human population. PMID:22589741

[Expression of carcinoembryonic antigen receptor in digestive organs].

PubMed

Zhao, Hui-min; Zhang, Sen; Gao, Feng

2010-08-01

To explore the significance of the expression of carcinoembryonic antigen receptors (CEAR) in digestive organs. Specimens were procured from 20 male BALB/c mice including esophagus, small intestine, stomach, colon, pancreas, and liver. Kupffer cells were obtained by density gradient centrifugation following enzymatic digestion of the fresh liver specimen. Immunohistochemistry and immunocytochemistry methods were used to detect CEAR in those organs or Kupffer cells. CEAR was found both in cytoplasm and nuclei of the digestive tract mucosal epithelial cells and pancreas islet cells, but only in the cytoplasm of liver cells, Kupffer cells, and smooth muscle cells of the whole digestive tract. The mean ranks of CEAR expression were 174.33 in the mucosal epithelial cells of colon, 160.70 in epithelial cells of small intestine, 139.18 in Kupffer cells, 137.43 in pancreas islet cells, 131.70 in liver cells, 124.23 in gastric epithelial cells, 77.15 in esophageal epithelial cells and 57.80-71.00 in smooth muscle cells of the entire digestive tract examined. There were significantly differences in the CEAR expression intensity among those positive cells (chi2=99.58, P<0.01) while CEAR was not present in submucosal connective tissue cells, pancreatic exocrine cells, or hepatic sinusoid endothelial cells. There are significantly differences in the expression of CEAR in the main digestive organs according to the different tissue and cells, which may play an important role in the carcinogenesis and hepatic metastasis from tumors of the digestive system.

Regulation of Plasmodium yoelii oocyst development by strain- and stage-specific small-subunit rRNA.

PubMed

Qi, Yanwei; Zhu, Feng; Eastman, Richard T; Fu, Young; Zilversmit, Martine; Pattaradilokrat, Sittiporn; Hong, Lingxian; Liu, Shengfa; McCutchan, Thomas F; Pan, Weiqing; Xu, Wenyue; Li, Jian; Huang, Fusheng; Su, Xin-zhuan

2015-03-10

One unique feature of malaria parasites is the differential transcription of structurally distinct rRNA (rRNA) genes at different developmental stages: the A-type genes are transcribed mainly in asexual stages, whereas the S-type genes are expressed mostly in sexual or mosquito stages. Conclusive functional evidence of different rRNAs in regulating stage-specific parasite development, however, is still absent. Here we performed genetic crosses of Plasmodium yoelii parasites with one parent having an oocyst development defect (ODD) phenotype and another producing normal oocysts to identify the gene(s) contributing to the ODD. The parent with ODD--characterized as having small oocysts and lacking infective sporozoites--was obtained after introduction of a plasmid with a green fluorescent protein gene into the parasite genome and subsequent passages in mice. Quantitative trait locus analysis of genome-wide microsatellite genotypes of 48 progeny from the crosses linked an ~200-kb segment on chromosome 6 containing one of the S-type genes (D-type small subunit rRNA gene [D-ssu]) to the ODD. Fine mapping of the plasmid integration site, gene expression pattern, and gene knockout experiments demonstrated that disruption of the D-ssu gene caused the ODD phenotype. Interestingly, introduction of the D-ssu gene into the same parasite strain (self), but not into a different subspecies, significantly affected or completely ablated oocyst development, suggesting a stage- and subspecies (strain)-specific regulation of oocyst development by D-ssu. This study demonstrates that P. yoelii D-ssu is essential for normal oocyst and sporozoite development and that variation in the D-ssu sequence can have dramatic effects on parasite development. Malaria parasites are the only known organisms that express structurally distinct rRNA genes at different developmental stages. The differential expression of these genes suggests that they play unique roles during the complex life cycle of the parasites. Conclusive functional proof of different rRNAs in regulating parasite development, however, is still absent or controversial. Here we functionally demonstrate for the first time that a stage-specifically expressed D-type small-subunit rRNA gene (D-ssu) is essential for oocyst development of the malaria parasite Plasmodium yoelii in the mosquito. This study also shows that variations in D-ssu sequence and/or the timing of transcription may have profound effects on parasite oocyst development. The results show that in addition to protein translation, rRNAs of malaria parasites also regulate parasite development and differentiation in a strain-specific manner, which can be explored for controlling parasite transmission. Copyright © 2015 Qi et al.

Using Peptide-Level Proteomics Data for Detecting Differentially Expressed Proteins.

PubMed

Suomi, Tomi; Corthals, Garry L; Nevalainen, Olli S; Elo, Laura L

2015-11-06

The expression of proteins can be quantified in high-throughput means using different types of mass spectrometers. In recent years, there have emerged label-free methods for determining protein abundance. Although the expression is initially measured at the peptide level, a common approach is to combine the peptide-level measurements into protein-level values before differential expression analysis. However, this simple combination is prone to inconsistencies between peptides and may lose valuable information. To this end, we introduce here a method for detecting differentially expressed proteins by combining peptide-level expression-change statistics. Using controlled spike-in experiments, we show that the approach of averaging peptide-level expression changes yields more accurate lists of differentially expressed proteins than does the conventional protein-level approach. This is particularly true when there are only few replicate samples or the differences between the sample groups are small. The proposed technique is implemented in the Bioconductor package PECA, and it can be downloaded from http://www.bioconductor.org.

Transcriptome responses to temperature, water availability and photoperiod are conserved among mature trees of two divergent Douglas-fir provenances from a coastal and an interior habitat.

PubMed

Hess, Moritz; Wildhagen, Henning; Junker, Laura Verena; Ensminger, Ingo

2016-08-26

Local adaptation and phenotypic plasticity are important components of plant responses to variations in environmental conditions. While local adaptation has been widely studied in trees, little is known about plasticity of gene expression in adult trees in response to ever changing environmental conditions in natural habitats. Here we investigate plasticity of gene expression in needle tissue between two Douglas-fir provenances represented by 25 adult trees using deep RNA sequencing (RNA-Seq). Using linear mixed models we investigated the effect of temperature, soil water availability and photoperiod on the abundance of 59189 detected transcripts. Expression of more than 80 % of all identified transcripts revealed a response to variations in environmental conditions in the field. GO term overrepresentation analysis revealed gene expression responses to temperature, soil water availability and photoperiod that are highly conserved among many plant taxa. However, expression differences between the two Douglas-fir provenances were rather small compared to the expression differences observed between individual trees. Although the effect of environment on global transcript expression was high, the observed genotype by environment (GxE) interaction of gene expression was surprisingly low, since only 21 of all detected transcripts showed a GxE interaction. The majority of the transcriptome responses in plant leaf tissue is driven by variations in environmental conditions. The small variation between individuals and populations suggests strong conservation of this response within Douglas-fir. Therefore we conclude that plastic transcriptome responses to variations in environmental conditions are only weakly affected by local adaptation in Douglas-fir.

Gene network-based analysis identifies two potential subtypes of small intestinal neuroendocrine tumors.

PubMed

Kidd, Mark; Modlin, Irvin M; Drozdov, Ignat

2014-07-15

Tumor transcriptomes contain information of critical value to understanding the different capacities of a cell at both a physiological and pathological level. In terms of clinical relevance, they provide information regarding the cellular "toolbox" e.g., pathways associated with malignancy and metastasis or drug dependency. Exploration of this resource can therefore be leveraged as a translational tool to better manage and assess neoplastic behavior. The availability of public genome-wide expression datasets, provide an opportunity to reassess neuroendocrine tumors at a more fundamental level. We hypothesized that stringent analysis of expression profiles as well as regulatory networks of the neoplastic cell would provide novel information that facilitates further delineation of the genomic basis of small intestinal neuroendocrine tumors. We re-analyzed two publically available small intestinal tumor transcriptomes using stringent quality control parameters and network-based approaches and validated expression of core secretory regulatory elements e.g., CPE, PCSK1, secretogranins, including genes involved in depolarization e.g., SCN3A, as well as transcription factors associated with neurodevelopment (NKX2-2, NeuroD1, INSM1) and glucose homeostasis (APLP1). The candidate metastasis-associated transcription factor, ST18, was highly expressed (>14-fold, p < 0.004). Genes previously associated with neoplasia, CEBPA and SDHD, were decreased in expression (-1.5 - -2, p < 0.02). Genomic interrogation indicated that intestinal tumors may consist of two different subtypes, serotonin-producing neoplasms and serotonin/substance P/tachykinin lesions. QPCR validation in an independent dataset (n = 13 neuroendocrine tumors), confirmed up-regulated expression of 87% of genes (13/15). An integrated cellular transcriptomic analysis of small intestinal neuroendocrine tumors identified that they are regulated at a developmental level, have key activation of hypoxic pathways (a known regulator of malignant stem cell phenotypes) as well as activation of genes involved in apoptosis and proliferation. Further refinement of these analyses by RNAseq studies of large-scale databases will enable definition of individual master regulators and facilitate the development of novel tissue and blood-based tools to better understand diagnose and treat tumors.

Identification of candidate miRNAs and expression profile of yak oocytes before and after in vitro maturation by high-throughput sequencing.

PubMed

Xiong, X R; Lan, D L; Li, J; Zi, X D; Li, M Y

2016-12-01

Small RNA represents several unique non-coding RNA classes that have important function in a wide range of biological processes including development of germ cells and early embryonic, cell differentiation, cell proliferation and apoptosis in diverse organisms. However, little is known about their expression profiles and effects in yak oocytes maturation and early development. To investigate the function of small RNAs in the maturation process of yak oocyte and early development, two small RNA libraries of oocytes were constructed from germinal vesicle stage (GV) and maturation in vitro to metaphase II-arrested stage (M II) and then sequenced using small RNA high-throughput sequencing technology. A total of 9,742,592 and 12,168,523 clean reads were obtained from GV and M II oocytes, respectively. In total, 801 and 1,018 known miRNAs were acquired from GV and M II oocytes, and 75 miRNAs were found to be significantly differentially expressed: 47 miRNAs were upregulated and 28 miRNAs were downregulated in the M II oocytes compared to the GV stage. Among the upregulated miRNAs, miR-342 has the largest fold change (9.25-fold). Six highly expressed miRNAs (let-7i, miR-10b, miR-10c, miR-143, miR-146b and miR-148) were validated by real-time quantitative PCR (RT-qPCR) and consistent with the sequencing results. Furthermore, the expression patterns of two miRNAs and their potential targets were analysed in different developmental stages of oocytes and early embryos. This study provides the first miRNA profile in the mature process of yak oocyte. Seventy-five miRNAs are expressed differentially in GV and M II oocytes as well as among different development stages of early embryos, suggesting miRNAs involved in regulating oocyte maturation and early development of yak. These results showed specific miRNAs in yak oocytes had dynamic changes during meiosis. Further functional and mechanistic studies on the miRNAs during meiosis may beneficial to understanding the role of miRNAs on meiotic division. © 2016 Blackwell Verlag GmbH.

Ambient temperature regulates the expression of a small set of sRNAs influencing plant development through NF-YA2 and YUC2.

PubMed

Gyula, Péter; Baksa, Ivett; Tóth, Tamás; Mohorianu, Irina; Dalmay, Tamás; Szittya, György

2018-06-01

Plants substantially alter their developmental program upon changes in the ambient temperature. The 21-24 nt small RNAs (sRNAs) are important gene expression regulators, which play a major role in development and adaptation. However, little is known about how the different sRNA classes respond to changes in the ambient temperature. We profiled the sRNA populations in four different tissues of Arabidopsis thaliana plants grown at 15, 21 and 27 °C. We found that only a small fraction (0.6%) of the sRNA loci are ambient temperature-controlled. We identified thermoresponsive miRNAs and identified their target genes using degradome libraries. We verified that the target of the thermoregulated miR169, NF-YA2, is also ambient temperature-regulated. NF-YA2, as the component of the conserved transcriptional regulator NF-Y complex, binds the promoter of the flowering time regulator FT and the auxin biosynthesis gene YUC2. Other differentially expressed loci include thermoresponsive phased siRNA loci that target various auxin pathway genes and tRNA fragments. Furthermore, a temperature dependent 24-nt heterochromatic siRNA locus in the promoter of YUC2 may contribute to the epigenetic regulation of auxin homeostasis. This holistic approach facilitated a better understanding of the role of different sRNA classes in ambient temperature adaptation of plants. This article is protected by copyright. All rights reserved.

The gut microbiota modulates host amino acid and glutathione metabolism in mice

PubMed Central

Mardinoglu, Adil; Shoaie, Saeed; Bergentall, Mattias; Ghaffari, Pouyan; Zhang, Cheng; Larsson, Erik; Bäckhed, Fredrik; Nielsen, Jens

2015-01-01

The gut microbiota has been proposed as an environmental factor that promotes the progression of metabolic diseases. Here, we investigated how the gut microbiota modulates the global metabolic differences in duodenum, jejunum, ileum, colon, liver, and two white adipose tissue depots obtained from conventionally raised (CONV-R) and germ-free (GF) mice using gene expression data and tissue-specific genome-scale metabolic models (GEMs). We created a generic mouse metabolic reaction (MMR) GEM, reconstructed 28 tissue-specific GEMs based on proteomics data, and manually curated GEMs for small intestine, colon, liver, and adipose tissues. We used these functional models to determine the global metabolic differences between CONV-R and GF mice. Based on gene expression data, we found that the gut microbiota affects the host amino acid (AA) metabolism, which leads to modifications in glutathione metabolism. To validate our predictions, we measured the level of AAs and N-acetylated AAs in the hepatic portal vein of CONV-R and GF mice. Finally, we simulated the metabolic differences between the small intestine of the CONV-R and GF mice accounting for the content of the diet and relative gene expression differences. Our analyses revealed that the gut microbiota influences host amino acid and glutathione metabolism in mice. PMID:26475342

Practical comparison of LC columns packed with different superficially porous particles for the separation of small molecules and medium size natural products.

PubMed

Yang, Peilin; McCabe, Terry; Pursch, Matthias

2011-11-01

Commercial C(18) columns packed with superficially porous particles of different sizes and shell thicknesses (Ascentis Express, Kinetex, and Poroshell 120) or sub-2-μm totally porous particles (Acquity BEH) were systematically compared using a small molecule mixture and a complex natural product mixture as text probes. Significant efficiency loss was observed on 2.1-mm id columns even with a low dispersion ultra-high pressure liquid chromatography system. The Kinetex 4.6-mm id column packed with 2.6-μm particles exhibited the best overall efficiency for small molecule separations and the Poroshell 120 column showed better performance for mid-size natural product analytes. The Kinetex 2.1-mm id column packed with 1.7-μm particles did not deliver the expected performance and the possible reasons besides extra column effect have been proved to be frictional heating effect and poor column packing quality. Different column retentivities and selectivities have been observed on the four C(18) columns of different brands for the natural product separation. Column batch-to-batch variability that has been previously observed on the Ascentis Express column was also observed on the Kinetex and Poroshell 120 column. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Thymidylate synthase (TS) gene expression in primary lung cancer patients: a large-scale study in Japanese population.

PubMed

Tanaka, F; Wada, H; Fukui, Y; Fukushima, M

2011-08-01

Previous small-sized studies showed lower thymidylate synthase (TS) expression in adenocarcinoma of the lung, which may explain higher antitumor activity of TS-inhibiting agents such as pemetrexed. To quantitatively measure TS gene expression in a large-scale Japanese population (n = 2621) with primary lung cancer, laser-captured microdissected sections were cut from primary tumors, surrounding normal lung tissues and involved nodes. TS gene expression level in primary tumor was significantly higher than that in normal lung tissue (mean TS/β-actin, 3.4 and 1.0, respectively; P < 0.01), and TS gene expression level was further higher in involved node (mean TS/β-actin, 7.7; P < 0.01). Analyses of TS gene expression levels in primary tumor according to histologic cell type revealed that small-cell carcinoma showed highest TS expression (mean TS/β-actin, 13.8) and that squamous cell carcinoma showed higher TS expression as compared with adenocarcinoma (mean TS/β-actin, 4.3 and 2.3, respectively; P < 0.01); TS gene expression was significantly increased along with a decrease in the grade of tumor cell differentiation. There was no significant difference in TS gene expression according to any other patient characteristics including tumor progression. Lower TS expression in adenocarcinoma of the lung was confirmed in a large-scale study.

Development, application, and sensitivity analysis of a water quality index for drinking water management in small systems.

PubMed

Scheili, A; Rodriguez, Manuel J; Sadiq, R

2015-11-01

The aim of this study was to produce a drinking water assessment tool for operators of small distribution systems. A drinking water quality index (DWQI) was developed and applied to small systems based on the water quality index of the Canadian Council of Ministers of Environment. The drinking water quality index was adapted to specific needs by creating four drinking water quality scenarios. First, the temporal and spatial dimensions of drinking water quality variability were taken into account. The DWQI was designed to express global drinking water quality according to different monitoring frequencies. Daily, monthly, and seasonal assessment was also considered. With the data made available, it was possible to use the index as a spatial monitoring tool and express water quality in different points in the distribution system. Moreover, adjustments were made to prioritize the type of contaminant to monitor. For instance, monitoring contaminants with acute health effects led to a scenario based on daily measures, including easily accessible and affordable water quality parameters. On the other hand, contaminants with chronic effects, especially disinfection by-products, were considered in a seasonal monitoring scenario where disinfection by-product reference values were redefined according to their seasonal variability. A sensitivity analysis was also carried out to validate the index. Globally, the DWQI developed is adapted to the needs of small systems. In fact, expressing drinking water quality using the DWQI contributes to the identification of problematic periods and segments in the distribution system. Further work may include this index in the development of a customized decision-making tool for small-system operators and managers.

Equivalent Electromagnetic Constants for Microwave Application to Composite Materials for the Multi-Scale Problem

PubMed Central

Fujisaki, Keisuke; Ikeda, Tomoyuki

2013-01-01

To connect different scale models in the multi-scale problem of microwave use, equivalent material constants were researched numerically by a three-dimensional electromagnetic field, taking into account eddy current and displacement current. A volume averaged method and a standing wave method were used to introduce the equivalent material constants; water particles and aluminum particles are used as composite materials. Consumed electrical power is used for the evaluation. Water particles have the same equivalent material constants for both methods; the same electrical power is obtained for both the precise model (micro-model) and the homogeneous model (macro-model). However, aluminum particles have dissimilar equivalent material constants for both methods; different electric power is obtained for both models. The varying electromagnetic phenomena are derived from the expression of eddy current. For small electrical conductivity such as water, the macro-current which flows in the macro-model and the micro-current which flows in the micro-model express the same electromagnetic phenomena. However, for large electrical conductivity such as aluminum, the macro-current and micro-current express different electromagnetic phenomena. The eddy current which is observed in the micro-model is not expressed by the macro-model. Therefore, the equivalent material constant derived from the volume averaged method and the standing wave method is applicable to water with a small electrical conductivity, although not applicable to aluminum with a large electrical conductivity. PMID:28788395

Retinal dehydrogenase gene expression in stomach and small intestine of rats during postnatal development and in vitamin A deficiency.

PubMed

Bhat, P V

1998-04-17

Retinal dehydrogenase (RALDH) catalyzes the oxidation of retinal to all-trans and 9-cis retinoic acid, which function as ligands controlling RAR and RXR nuclear receptor-signaling pathways. We have recently shown the expression of RALDH transcript in the stomach and small intestine by reverse transcription polymerase chain reaction [Bhat, P.V., Labrecque J., Dumas, F., Lacroix, A. and Yoshida, A. (1995) Gene 166, 303-306]. We have examined RALDH expression in the stomach and small intestine before and during postnatal development and in vitamin A deficiency by assaying for mRNA levels and protein as well as for enzyme activity. In -2 day fetuses, RALDH expression was high in the small intestine, whereas RALDH protein was not detectable in the stomach. However, expression of RALDH was seen in the stomach after birth, and gradually increased with age and reached the highest level at postnatal day 42. In the intestine, RALDH expression decreased postnatally. Vitamin A deficiency up-regulated RALDH expression in the stomach and small intestine, and administration of retinoids down-regulated the RALDH expression in these tissues. These results show the differential expression of RALDH in the stomach and small intestine during postnatal development, and that vitamin A status regulates the expression of RALDH gene in these tissues.

Age-Related Gene Expression Differences in Monocytes from Human Neonates, Young Adults, and Older Adults

PubMed Central

Tong, Ann-Jay; Kollmann, Tobias R.; Smale, Stephen T.

2015-01-01

A variety of age-related differences in the innate and adaptive immune systems have been proposed to contribute to the increased susceptibility to infection of human neonates and older adults. The emergence of RNA sequencing (RNA-seq) provides an opportunity to obtain an unbiased, comprehensive, and quantitative view of gene expression differences in defined cell types from different age groups. An examination of ex vivo human monocyte responses to lipopolysaccharide stimulation or Listeria monocytogenes infection by RNA-seq revealed extensive similarities between neonates, young adults, and older adults, with an unexpectedly small number of genes exhibiting statistically significant age-dependent differences. By examining the differentially induced genes in the context of transcription factor binding motifs and RNA-seq data sets from mutant mouse strains, a previously described deficiency in interferon response factor-3 activity could be implicated in most of the differences between newborns and young adults. Contrary to these observations, older adults exhibited elevated expression of inflammatory genes at baseline, yet the responses following stimulation correlated more closely with those observed in younger adults. Notably, major differences in the expression of constitutively expressed genes were not observed, suggesting that the age-related differences are driven by environmental influences rather than cell-autonomous differences in monocyte development. PMID:26147648

[Analysis of tissue-specific differentially methylated genes with differential gene expression in non-small cell lung cancer].

PubMed

Yin, L G; Zou, Z Q; Zhao, H Y; Zhang, C L; Shen, J G; Qi, L; Qi, M; Xue, Z Q

2014-01-01

Adenocarcinoma (ADC) and squamous cell carcinomas (SCC) are two subtypes of non-small cell lung carcinomas which are regarded as the leading cause of cancer-related malignancy worldwide. The aim of this study is to detect the differentially methylated loci (DMLs) and differentially methylated genes (DMGs) of these two tumor sets, and then to illustrate the different expression level of specific methylated genes. Using TCGA database and Illumina HumanMethylation 27 arrays, we first screened the DMGs and DMLs in tumor samples. Then, we explored the BiologicalProcess terms of hypermethylated and hypomethylated genes using Functional Gene Ontology (GO) catalogues. Hypermethylation intensively occurred in CpG-island, whereas hypomethylation was located in non-CpG-island. Most SCC and ADC hypermethylated genes involved GO function of DNA dependenit regulation of transcription, and hypomethylated genes mainly 'enriched in the term of immune responses. Additionally, the expression level of specific differentially methylated genesis distinctbetween ADC and SCC. It is concluded that ADC and SCC have different methylated status that might play an important role in carcinogenesis.

Genome-wide analysis and expression profiling of the Solanum tuberosum aquaporins.

PubMed

Venkatesh, Jelli; Yu, Jae-Woong; Park, Se Won

2013-12-01

Aquaporins belongs to the major intrinsic proteins involved in the transcellular membrane transport of water and other small solutes. A comprehensive genome-wide search for the homologues of Solanum tuberosum major intrinsic protein (MIP) revealed 41 full-length potato aquaporin genes. All potato aquaporins are grouped into five subfamilies; plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), NOD26-like intrinsic proteins (NIPs), small basic intrinsic proteins (SIPs) and x-intrinsic proteins (XIPs). Functional predictions based on the aromatic/arginine (ar/R) selectivity filters and Froger's positions showed a remarkable difference in substrate transport specificity among subfamilies. The expression pattern of potato aquaporins, examined by qPCR analysis, showed distinct expression profiles in various organs and tuber developmental stages. Furthermore, qPCR analysis of potato plantlets, subjected to various abiotic stresses revealed the marked effect of stresses on expression levels of aquaporins. Taken together, the expression profiles of aquaporins imply that aquaporins play important roles in plant growth and development, in addition to maintaining water homeostasis in response to environmental stresses. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Cell surface expression of CCR5 and other host factors influence the inhibition of HIV-1 infection of human lymphocytes by CCR5 ligands

PubMed Central

Ketas, Thomas J.; Kuhmann, Shawn E.; Palmer, Ashley; Zurita, Juan; He, Weijing; Ahuja, Sunil K.; Klasse, Per Johan; Moore, John P.

2007-01-01

Several CCR5 ligands, including small molecules and monoclonal antibodies (MAbs), are being developed as therapies for infection with strains of human immunodeficiency virus type 1 (HIV-1) that use CCR5 for entry (R5 viruses). The efficacy of such therapies could be influenced by inter-individual differences in host factors, such as CCR5 expression levels. To study this, we used peripheral blood mononuclear cells (PBMCs) from humans and rhesus macaques. The half-maximal inhibitory concentrations (IC50) of the small-molecule CCR5 ligands CMPD167, UK427,857 and SCH-D, and of the PRO 140 MAb, differ by >2 logs in a donor-dependent manner. We studied this variation by using flow cytometry to measure CCR5 expression on PBMCs from six of the human donors: the IC50 values of both SCH-D and PRO 140 correlated with CCR5 expression (R2 = 0.64 and 0.99, respectively). We also determined the efficacy of the CCR5 ligands against HIV-1 infection of HeLa-derived cell lines that express CD4 at the same level but vary 2-fold in CCR5 expression (JC.48 and JC.53 cells). The moderately greater CCR5 expression on the JC.53 than the JC.48 cells was associated with proportionately higher median IC50 values for all four CCR5 ligands but not for a soluble CD4-based inhibitor or a non-nucleoside reverse transcriptase inhibitor. We conclude that differences in CCR5 expression on human PBMCs, which can be affected by CCL3L1 gene dose, may influence the antiviral potency of CCR5 ligands in vitro, but other host factors are also likely to be involved. These host factors may affect the clinical activity of CCR5 inhibitors, including their use as topical microbicides to prevent HIV-1 transmission. PMID:17428518

Expression and regulation of anti-mullerian hormone in an oviparous species, the hen.

PubMed

Johnson, P A; Kent, T R; Urick, M E; Giles, J R

2008-01-01

Anti-mullerian hormone (AMH) has a critical role in regression of the mullerian duct system during development in male mammalian and avian species and in regression of the right oviduct in female avian species. AMH in adult female birds has not been investigated. Chicken-specific cDNA primers were used to isolate Amh by RT-PCR. This probe was used in Northern blot analysis to identify a 2.8-kb band with expression in total ovarian RNA and in granulosa cell RNA. Quantitative real-time PCR was used to assess Amh expression in follicles of different maturity (1, 3, 5, and 6-12 mm and the largest F1 follicle; n = 4-6 of each size). There was an increased amount of Amh mRNA in the granulosa layer of the smaller follicles and a lower amount in the granulosa layer of the larger follicles (P < 0.01). There was no difference in granulosa Amh expression between the germinal disc and non-germinal disc region of 6- to 12-mm follicles, although expression differed with follicle size (P < 0.01). To examine hormone regulation of Amh, granulosa cells (from 6- to 8-mm follicles) were cultured with various concentrations of estradiol (E(2)) and progesterone (P(4)), and Amh mRNA was assessed. Neither E(2) nor P(4) influenced Amh mRNA accumulation. Granulosa cells were also cultured in the presence of oocyte-conditioned medium (OCM), which decreased Amh mRNA expression in a dose-related manner (P < 0.05); FSH receptor expression was not affected. Heat treatment of OCM abolished the effect, but growth differentiation factor 9 antiserum did not block the suppression. Immunohistochemistry confirmed that the granulosa layer was the predominant source of AMH in the small follicles of the hen and indicated that AMH was present early in follicle development, with expression in very small follicles (approximately 150 mum).

Chemotherapy treatment is associated with altered PD-L1 expression in lung cancer patients.

PubMed

Rojkó, Lívia; Reiniger, Lilla; Téglási, Vanda; Fábián, Katalin; Pipek, Orsolya; Vágvölgyi, Attila; Agócs, László; Fillinger, János; Kajdácsi, Zita; Tímár, József; Döme, Balázs; Szállási, Zoltán; Moldvay, Judit

2018-04-19

While the predictive value of programmed cell death ligand-1 (PD-L1) protein expression for immune checkpoint inhibitor therapy of lung cancer has been extensively studied, the impact of standard platinum-based chemotherapy on PD-L1 or programmed cell death-1 (PD-1) expression is unknown. The aim of this study was to determine the changes in PD-L1 expression of tumor cells (TC) and immune cells (IC), in PD-1 expression of IC, and in the amount of stromal mononuclear cell infiltration after platinum-based chemotherapy in patients with lung cancer. We determined the amount of stromal mononuclear cells and PD-L1/PD-1 expressions by immunohistochemistry in bronchoscopic biopsy samples including 20 adenocarcinomas (ADC), 15 squamous cell carcinomas (SCC), 2 other types of non-small cell lung cancer, and 4 small cell lung cancers together with their corresponding surgical resection tissues after platinum-based chemotherapy. PD-L1 expression of TC decreased in ten patients (24.4%) and increased in three patients (7.32%) after neoadjuvant chemotherapy (p = 0.051). The decrease in PD-L1 expression, however, was significant only in patients who received cisplatin-gemcitabine combination (p = 0.020), while in the carboplatin-paclitaxel group, no similar tendency could be observed (p = 0.432). There was no difference between ADC and SCC groups. Neither PD-1 expression nor the amount of stromal IC infiltration showed significant changes after chemotherapy. This is the first study, in which both PD-L1 and PD-1 expression were analyzed together with the amount of stromal IC infiltration in different histological subtypes of lung cancer before and after platinum-based chemotherapy. Our results confirm that chemotherapy decreases PD-L1 expression of TC in a subset of patients, therefore, rebiopsy and re-evaluation of PD-L1 expression may be necessary for the indication of immune checkpoint inhibitor therapy.

Small Molecule-Induced Complement Factor D (Adipsin) Promotes Lipid Accumulation and Adipocyte Differentiation

PubMed Central

Jang, Byung-Hyun; Chang, Seo-Hyuk; Yun, Ui Jeong; Park, Ki-Moon; Waki, Hironori; Li, Dean Y.; Tontonoz, Peter; Park, Kye Won

2016-01-01

Adipocytes are differentiated by various transcriptional cascades integrated on the master regulator, Pparγ. To discover new genes involved in adipocyte differentiation, preadipocytes were treated with three newly identified pro-adipogenic small molecules and GW7845 (a Pparγ agonist) for 24 hours and transcriptional profiling was analyzed. Four genes, Peroxisome proliferator-activated receptor γ (Pparγ), human complement factor D homolog (Cfd), Chemokine (C-C motif) ligand 9 (Ccl9), and GIPC PDZ Domain Containing Family Member 2 (Gipc2) were induced by at least two different small molecules but not by GW7845. Cfd and Ccl9 expressions were specific to adipocytes and they were altered in obese mice. Small hairpin RNA (shRNA) mediated knockdown of Cfd in preadipocytes inhibited lipid accumulation and expression of adipocyte markers during adipocyte differentiation. Overexpression of Cfd promoted adipocyte differentiation, increased C3a production, and led to induction of C3a receptor (C3aR) target gene expression. Similarly, treatments with C3a or C3aR agonist (C4494) also promoted adipogenesis. C3aR knockdown suppressed adipogenesis and impaired the pro-adipogenic effects of Cfd, further suggesting the necessity for C3aR signaling in Cfd-mediated pro-adipogenic axis. Together, these data show the action of Cfd in adipogenesis and underscore the application of small molecules to identify genes in adipocytes. PMID:27611793

Analysis of myosin heavy chain mRNA expression by RT-PCR

NASA Technical Reports Server (NTRS)

Wright, C.; Haddad, F.; Qin, A. X.; Baldwin, K. M.

1997-01-01

An assay was developed for rapid and sensitive analysis of myosin heavy chain (MHC) mRNA expression in rodent skeletal muscle. Only 2 microg of total RNA were necessary for the simultaneous analysis of relative mRNA expression of six different MHC genes. We designed synthetic DNA fragments as internal standards, which contained the relevant primer sequences for the adult MHC mRNAs type I, IIa, IIx, IIb as well as the embryonic and neonatal MHC mRNAs. A known amount of the synthetic fragment was added to each polymerase chain reaction (PCR) and yielded a product of different size than the amplified MHC mRNA fragment. The ratio of amplified MHC fragment to synthetic fragment allowed us to calculate percentages of the gene expression of the different MHC genes in a given muscle sample. Comparison with the traditional Northern blot analysis demonstrated that our reverse transcriptase-PCR-based assay was reliable, fast, and quantitative over a wide range of relative MHC mRNA expression in a spectrum of adult and neonatal rat skeletal muscles. Furthermore, the high sensitivity of the assay made it very useful when only small quantities of tissue were available. Statistical analysis of the signals for each MHC isoform across the analyzed samples showed a highly significant correlation between the PCR and the Northern signals as Pearson correlation coefficients ranged between 0.77 and 0.96 (P < 0.005). This assay has potential use in analyzing small muscle samples such as biopsies and samples from pre- and/or neonatal stages of development.

Genomic dissection of small RNAs in wild rice (Oryza rufipogon): lessons for rice domestication.

PubMed

Wang, Yu; Bai, Xuefei; Yan, Chenghai; Gui, Yiejie; Wei, Xinghua; Zhu, Qian-Hao; Guo, Longbiao; Fan, Longjiang

2012-11-01

The lack of a MIRNA set and genome sequence of wild rice (Oryza rufipogon) has prevented us from determining the role of MIRNA genes in rice domestication. In this study, a genome, three small RNA populations and a degradome of O. rufipogon were sequenced by Illumina platform and the expression levels of microRNAs (miRNAs) were investigated by miRNA chips. A de novo O. rufipogon genome was assembled using c. 55× coverage of raw sequencing data and a total of 387 MIRNAs were identified in the O. rufipogon genome based on c. 5.2 million unique small RNA reads from three different tissues of O. rufipogon. Of these, O. rufipogon MIRNAs, 259 were not found in the cultivated rice, suggesting a loss of these MIRNAs in the cultivated rice. We also found that 48 MIRNAs were novel in the cultivated rice, suggesting that they were potential targets of domestication selection. Some miRNAs showed significant expression differences between wild and cultivated rice, suggesting that expression of miRNA could also be a target of domestication, as demonstrated for the miR164 family. Our results illustrated that MIRNA genes, like protein-coding genes, might have been significantly shaped during rice domestication and could be one of the driving forces that contributed to rice domestication. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

Aquaporin family genes exhibit developmentally-regulated and host-dependent transcription patterns in the sea louse Caligus rogercresseyi.

PubMed

Farlora, Rodolfo; Valenzuela-Muñoz, Valentina; Chávez-Mardones, Jacqueline; Gallardo-Escárate, Cristian

2016-07-01

Aquaporins are small integral membrane proteins that function as pore channels for the transport of water and other small solutes across the cell membrane. Considering the important roles of these proteins in several biological processes, including host-parasite interactions, there has been increased research on aquaporin proteins recently. The present study expands on the knowledge of aquaporin family genes in parasitic copepods, examining diversity and expression during the ontogeny of the sea louse Caligus rogercresseyi. Furthermore, aquaporin expression was evaluated during the early infestation of Atlantic (Salmo salar) and Coho salmon (Oncorhynchus kisutch). Deep transcriptome sequencing data revealed eight full length and two partial open reading frames belonging to the aquaporin protein family. Clustering analyses with identified Caligidae sequences revealed three major clades of aquaglyceroporins (Cr-Glp), classical aquaporin channels (Cr-Bib and Cr-PripL), and unorthodox aquaporins (Cr-Aqp12-like). In silico analysis revealed differential expression of aquaporin genes between developmental stages and between sexes. Male-biased expression of Cr-Glp1_v1 and female-biased expression of Cr-Bib were further confirmed in adults by RT-qPCR. Additionally, gene expressions were measured for seven aquaporins during the early infestation stage. The majority of aquaporin genes showed significant differential transcription expressions between sea lice parasitizing different hosts, with Atlantic salmon sea lice exhibiting overall reduced expression as compared to Coho salmon. The observed differences in the regulation of aquaporin genes may reveal osmoregulatory adaptations associated with nutrient ingestion and metabolite waste export, exposing complex host-parasite relationships in C. rogercresseyi. Copyright © 2016 Elsevier B.V. All rights reserved.

Expression and regulation of the neutral amino acid transporter B0AT1 in rat small intestine

PubMed Central

Jando, Julia; Camargo, Simone M. R.; Herzog, Brigitte

2017-01-01

Absorption of neutral amino acids across the luminal membrane of intestinal enterocytes is mediated by the broad neutral amino acid transporter B0AT1 (SLC6A19). Its intestinal expression depends on co-expression of the membrane-anchored peptidase angiotensin converting enzyme 2 (ACE2) and is additionally enhanced by aminopeptidase N (CD13). We investigated in this study the expression of B0AT1 and its auxiliary peptidases as well as its transport function along the rat small intestine. Additionally, we tested its possible short- and long-term regulation by dietary proteins and amino acids. We showed by immunofluorescence that B0AT1, ACE2 and CD13 co-localize on the luminal membrane of small intestinal villi and by Western blotting that their protein expression increases in distal direction. Furthermore, we observed an elevated transport activity of the neutral amino acid L-isoleucine during the nocturnal active phase compared to the inactive one. Gastric emptying was delayed by intragastric application of an amino acid cocktail but we observed no acute dietary regulation of B0AT1 protein expression and L-isoleucine transport. Investigation of the chronic dietary regulation of B0AT1, ACE2 and CD13 by different diets revealed an increased B0AT1 protein expression under amino acid-supplemented diet in the proximal section but not in the distal one and for ACE2 protein expression a reverse localization of the effect. Dietary regulation for CD13 protein expression was not as distinct as for the two other proteins. Ring uptake experiments showed a tendency for increased L-isoleucine uptake under amino acid-supplemented diet and in vivo L-isoleucine absorption was more efficient under high protein and amino acid-supplemented diet. Additionally, plasma levels of branched-chain amino acids were elevated under high protein and amino acid diet. Taken together, our experiments did not reveal an acute amino acid-induced regulation of B0AT1 but revealed a chronic dietary adaptation mainly restricted to the proximal segment of the small intestine. PMID:28915252

Different sucrose-isomaltase response of Caco-2 cells to glucose and maltose suggests dietary maltose sensing

USDA-ARS?s Scientific Manuscript database

Using the small intestine enterocyte Caco-2 cell model, sucrase-isomaltase (SI, the mucosal alpha-glucosidase complex) expression and modification were examined relative to exposure to different mono- and disaccharide glycemic carbohydrates. Caco-2/TC7 cells were grown on porous supports to post-con...

Overexpression of Pokemon in non-small cell lung cancer and foreshowing tumor biological behavior as well as clinical results.

PubMed

Zhao, Zhi-Hong; Wang, Sheng-Fa; Yu, Liang; Wang, Ju; Chang, Hao; Yan, Wei-Li; Zhang, Jian; Fu, Kai

2008-10-01

Transcription factor Pokemon, a central regulation gene of the important tumor suppressor alternative reading frame (ARF), exerted its activity by acting upstream of many tumor-suppressing genes and proto-oncogenes. Its expression in non-small cell lung cancer (NSCLC) and its clinical significance remains unclear. The aim of this study was to investigate the expression of Pokemon in non-small cell lung cancer and to explore its correlation with the clinical pathological characteristics and its influence on patients' prognosis. Observe the expression of Pokemon in NSCLC and investigate its mechanism and clinical significance. Determine the expression of Pokemon in human NSCLC cell lines as well as 55 cases of NSCLC tumor tissues, tumor adjacent tissues and surrounding tissues by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, and analyze the relationship between Pokemon expression in NSCLC tumor tissues and clinicopathological features. Determine 62 NSCLC tumor tissues (5 years ago) and p14(ARF) expression with immunohistochemical technique, discuss the correlation between them and assess the effect of Pokemon on prognosis of patients with lung cancer. Pokemon mRNA and protein took on high expression in lung cancer cell lines, and the expression difference between cancer tissues, tumor adjacent tissues and surrounding tissues had statistical significance (P<0.05). Pokemon expression and p14(ARF) expression were negatively correlated (r=-0.287). The expression of Pokemon was determined not to be associated with the patient's sex, age, smoking condition, tumor differentiation degree, histology and lymph node metastasis condition. However, its relationship with TNM staging was established (P<0.05). Furthermore, it was shown that the survival rate of patients with negative Pokemon expression was significantly higher than that of those with positive Pokemon expression (P=0.004), therefore, the expression of Pokemon is believed to be an independent factor affecting prognosis (P=0.034). There was high expression of Pokemon in NSCLC, Pokemon exerted carcinogenesis by inhibiting ARF and had some clinical significance for prognostic evaluation of the patients with NSCLC.

Human snoRNA-93 is processed into a microRNA-like RNA that promotes breast cancer cell invasion.

PubMed

Patterson, Dillon G; Roberts, Justin T; King, Valeria M; Houserova, Dominika; Barnhill, Emmaline C; Crucello, Aline; Polska, Caroline J; Brantley, Lucas W; Kaufman, Garrett C; Nguyen, Michael; Santana, Megann W; Schiller, Ian A; Spicciani, Julius S; Zapata, Anastasia K; Miller, Molly M; Sherman, Timothy D; Ma, Ruixia; Zhao, Hongyou; Arora, Ritu; Coley, Alexander B; Zeidan, Melody M; Tan, Ming; Xi, Yaguang; Borchert, Glen M

2017-01-01

Genetic searches for tumor suppressors have recently linked small nucleolar RNA misregulations with tumorigenesis. In addition to their classically defined functions, several small nucleolar RNAs are now known to be processed into short microRNA-like fragments called small nucleolar RNA-derived RNAs. To determine if any small nucleolar RNA-derived RNAs contribute to breast malignancy, we recently performed a RNA-seq-based comparison of the small nucleolar RNA-derived RNAs of two breast cancer cell lines (MCF-7 and MDA-MB-231) and identified small nucleolar RNA-derived RNAs derived from 13 small nucleolar RNAs overexpressed in MDA-MB-231s. Importantly, we find that inhibiting the most differentially expressed of these small nucleolar RNA-derived RNAs (sdRNA-93) in MDA-MB-231 cells results primarily in a loss of invasiveness, whereas increased sdRNA-93 expression in either cell line conversely results in strikingly enhanced invasion. Excitingly, we recently determined sdRNA-93 expressions in small RNA-seq data corresponding to 116 patient tumors and normal breast controls, and while we find little sdRNA-93 expression in any of the controls and only sporadic expression in most subtypes, we find robust expression of sdRNA-93 in 92.8% of Luminal B Her2+tumors. Of note, our analyses also indicate that at least one of sdRNA-93's endogenous roles is to regulate the expression of Pipox, a sarcosine metabolism-related protein whose expression significantly correlates with distinct molecular subtypes of breast cancer. We find sdRNA-93 can regulate the Pipox 3'UTR via standard reporter assays and that manipulating endogenous sdRNA-93 levels inversely correlates with altered Pipox expression. In summary, our results strongly indicate that sdRNA-93 expression actively contributes to the malignant phenotype of breast cancer through participating in microRNA-like regulation.

Protein disorder is positively correlated with gene expression in E. coli

PubMed Central

Paliy, Oleg; Gargac, Shawn M.; Cheng, Yugong; Uversky, Vladimir N.; Dunker, A. Keith

2009-01-01

We considered on a global scale the relationship between the predicted fraction of protein disorder and RNA and protein expression in E. coli. Fraction of protein disorder correlated positively with both measured RNA expression levels of E. coli genes in three different growth media and with predicted abundance levels of E. coli proteins. Though weak, the correlation was highly significant. Correlation of protein disorder with RNA expression did not depend on the growth rate of E. coli cultures and was not caused by a small subset of genes showing exceptionally high concordance in their disorder and expression levels. Global analysis was complemented by detailed consideration of several groups of proteins. PMID:18465893

High-throughput characterization of Echinococcus spp. metacestode miRNomes.

PubMed

Cucher, Marcela; Macchiaroli, Natalia; Kamenetzky, Laura; Maldonado, Lucas; Brehm, Klaus; Rosenzvit, Mara Cecilia

2015-03-01

Echinococcosis is a worldwide zoonosis of great public health concern, considered a neglected disease by the World Health Organisation. The cestode parasites Echinococcus granulosus sensu lato (s. l.) and Echinococcus multilocularis are the main aetiological agents. In the intermediate host, these parasites display particular developmental traits that lead to different patterns of disease progression. In an attempt to understand the causes of these differences, we focused on the analysis of microRNAs (miRNAs), small non-coding regulatory RNAs with major roles in development of animals and plants. In this work, we analysed the small RNA expression pattern of the metacestode, the stage of sanitary relevance, and provide a detailed description of Echinococcus miRNAs. Using high-throughput small RNA sequencing, we believe that we have carried out the first experimental identification of miRNAs in E. multilocularis and have expanded the Echinococcus miRNA catalogue to 38 miRNA genes, including one miRNA only present in E. granulosus s. l. Our findings show that although both species share the top five highest expressed miRNAs, 13 are differentially expressed, which could be related to developmental differences. We also provide evidence that uridylation is the main miRNA processing mechanism in Echinococcus spp. These results provide detailed information on Echinococcus miRNAs, which is the first step in understanding their role in parasite biology and disease establishment and/or progression, and their future potential use as drug or diagnostic targets. Copyright © 2015 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Small RNA sorting: matchmaking for Argonautes

PubMed Central

Czech, Benjamin; Hannon, Gregory J.

2013-01-01

Small RNAs directly or indirectly impact nearly every biological process in eukaryotic cells. To perform their myriad roles, not only must precise small RNA species be generated, but they must also be loaded into specific effector complexes called RNA-induced silencing complexes (RISCs). Argonaute proteins form the core of RISCs and different members of this large family have specific expression patterns, protein binding partners and biochemical capabilities. In this Review, we explore the mechanisms that pair specific small RNA strands with their partner proteins, with an eye towards the substantial progress that has been recently made in understanding the sorting of the major small RNA classes — microRNAs (miRNAs) and small interfering RNAs (siRNAs) — in plants and animals. PMID:21116305

Data on enhanced expression and purification of camelid single domain antibodies from Escherichia coli classical inclusion bodies.

PubMed

Maggi, Maristella; Scotti, Claudia

2017-06-01

Heterologous expression of high amounts of recombinant proteins is a milestone for research and industrial purposes. Single domain antibodies (sdAbs) are heavy-chain only antibody fragments with applications in the biotechnological, medical and industrial fields. The simple nature and small size of sdAbs allows for efficient expression of the soluble molecule in different hosts. However, in some cases, it results in low functional protein yield. To overcome this limitation, expression of a 6xHistag sdAb was attempted in different conditions in Escherichia coli BL21(DE3) cells. Data showed that high amount of sdAb can be expressed in E. coli classical inclusion bodies, efficiently extracted by urea in a short-time, and properly purified by metal ion affinity chromatography. These data originate from the research article "Enhanced expression and purification of camelid single domain VHH antibodies from classical inclusion bodies" Maggi and Scotti (2017) [1] (DOI: http://dx.doi.org/10.1016/j.pep.2017.02.007).

Potential proteins targeted by let-7f-5p in HeLa cells.

PubMed

Wang, Yu; Chen, Xiujuan; Zhang, Yi; Song, Jiandong

2017-07-24

MicroRNAs are a class of small, endogenous, non-coding RNAs mediating posttranscriptional gene silencing. The current authors hypothesized that let-7f-5p is likely involved in cell invasion and proliferation by regulating the expression of target genes. The current study combined let-7f-5p with iTRAQ to assess its effect on gene expression in HeLa cells. Results indicated that 164 proteins were expressed at different levels in HeLa cells overexpressing let-7f-5p and negative controls and that 172 proteins were expressed at different levels in let-7f-5p-silenced HeLa cells and negative controls. Results indicated that let-7f-5p may suppress insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) in HeLa cells.

The Use of EST Expression Matrixes for the Quality Control of Gene Expression Data

PubMed Central

Milnthorpe, Andrew T.; Soloviev, Mikhail

2012-01-01

EST expression profiling provides an attractive tool for studying differential gene expression, but cDNA libraries' origins and EST data quality are not always known or reported. Libraries may originate from pooled or mixed tissues; EST clustering, EST counts, library annotations and analysis algorithms may contain errors. Traditional data analysis methods, including research into tissue-specific gene expression, assume EST counts to be correct and libraries to be correctly annotated, which is not always the case. Therefore, a method capable of assessing the quality of expression data based on that data alone would be invaluable for assessing the quality of EST data and determining their suitability for mRNA expression analysis. Here we report an approach to the selection of a small generic subset of 244 UniGene clusters suitable for identification of the tissue of origin for EST libraries and quality control of the expression data using EST expression information alone. We created a small expression matrix of UniGene IDs using two rounds of selection followed by two rounds of optimisation. Our selection procedures differ from traditional approaches to finding “tissue-specific” genes and our matrix yields consistency high positive correlation values for libraries with confirmed tissues of origin and can be applied for tissue typing and quality control of libraries as small as just a few hundred total ESTs. Furthermore, we can pick up tissue correlations between related tissues e.g. brain and peripheral nervous tissue, heart and muscle tissues and identify tissue origins for a few libraries of uncharacterised tissue identity. It was possible to confirm tissue identity for some libraries which have been derived from cancer tissues or have been normalised. Tissue matching is affected strongly by cancer progression or library normalisation and our approach may potentially be applied for elucidating the stage of normalisation in normalised libraries or for cancer staging. PMID:22412959

Gene expression profiles of changes underlying different-sized human rotator cuff tendon tears.

PubMed

Chaudhury, Salma; Xia, Zhidao; Thakkar, Dipti; Hakimi, Osnat; Carr, Andrew J

2016-10-01

Progressive cellular and extracellular matrix (ECM) changes related to age and disease severity have been demonstrated in rotator cuff tendon tears. Larger rotator cuff tears demonstrate structural abnormalities that potentially adversely influence healing potential. This study aimed to gain greater insight into the relationship of pathologic changes to tear size by analyzing gene expression profiles from normal rotator cuff tendons, small rotator cuff tears, and large rotator cuff tears. We analyzed gene expression profiles of 28 human rotator cuff tendons using microarrays representing the entire genome; 11 large and 5 small torn rotator cuff tendon specimens were obtained intraoperatively from tear edges, which we compared with 12 age-matched normal controls. We performed real-time polymerase chain reaction and immunohistochemistry for validation. Torn rotator cuff tendons demonstrated upregulation of a number of key genes, such as matrix metalloproteinase 3, 10, 12, 13, 15, 21, and 25; a disintegrin and metalloproteinase (ADAM) 12, 15, and 22; and aggrecan. Amyloid was downregulated in all tears. Small tears displayed upregulation of bone morphogenetic protein 5. Chemokines and cytokines that may play a role in chemotaxis were altered; interleukins 3, 10, 13, and 15 were upregulated in tears, whereas interleukins 1, 8, 11, 18, and 27 were downregulated. The gene expression profiles of normal controls and small and large rotator cuff tear groups differ significantly. Extracellular matrix remodeling genes were found to contribute to rotator cuff tear pathogenesis. Rotator cuff tears displayed upregulation of a number of matrix metalloproteinase (3, 10, 12, 13, 15, 21, and 25), a disintegrin and metalloproteinase (ADAM 12, 15, and 22) genes, and downregulation of some interleukins (1, 8, and 27), which play important roles in chemotaxis. These gene products may potentially have a role as biomarkers of failure of healing or therapeutic targets to improve tendon healing. Copyright © 2016 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.

Transcriptome analysis reveals regional and temporal differences in mucosal immune system development in the small intestine of neonatal calves.

PubMed

Liang, Guanxiang; Malmuthuge, Nilusha; Bao, Hua; Stothard, Paul; Griebel, Philip J; Guan, Le Luo

2016-08-11

Postnatal development of the mammalian mucosal immune system is crucial for responding to the rapid colonization by commensal bacteria and possible exposure to pathogens. This study analyzed expression patterns for mRNAs and their relationship with microRNAs (miRNAs) in the bovine small intestine during the critical neonatal period (0 to 42 days). This analysis revealed molecular mechanisms regulating the postnatal development of the intestinal mucosal immune system. Small intestine samples (jejunum and ileum) were collected from newborn male, Holstein calves immediately post-partum (n = 3) and at 7 (n = 5), 21 (n = 5), and 42 (n = 5) days of age and the transcriptomes were profiled using RNA-Seq. When analyzing all time points collectively, greater expression of genes encoding the complement functional pathway, as well as lower expression of genes encoding Toll-like receptors and NOD-like receptors were observed in the jejunum when compared to the ileum. In addition, significant changes in the expression of immune-related genes were detected within the first week post-partum in both jejunum and ileum. For example, increased expression of genes encoding tight junction proteins (claudin 1, claudin 4 and occludin), an antimicrobial peptide (Regenerating Islet-Derived 3-γ), NOD-like receptors (NACHT, LRR and PYD domain-containing protein 3), regulatory T cell marker (forkhead box P3), and both anti-inflammatory (interleukin 10) and pro-inflammatory (interleukin 8) cytokines was observed throughout the small intestine of 7-day-old calves when compared to newborn calves. Moreover, the expression of mucosal immune-related genes were either positively or negatively correlated with total bacterial population depending on both intestinal region and age. The integrated analysis of miRNAs and mRNAs supported the conclusion that miRNAs may regulate temporal changes in the expression of genes encoding tight junction proteins (miR-335), cytokines (miR-335) and bacterial recognition (miR-100) during the first week of small intestine development. The rapid development of transcriptional differences between jejunum and ileum reveal that these two intestinal regions make distinct contributions to the intestinal mucosal immune system during the early neonatal period. In addition, transcriptome analysis indicates that the first week after birth is a very dynamic developmental period for the intestinal mucosal immune system and these changes may be regulated by both miRNAs and microbial colonization. Findings from this study indicate that a detailed analysis of both the abundance and diversity of the colonizing microbiome may be necessary to understand factors regulating the rapid development of the mucosal immune system during the first week of life.

75 FR 80561 - Community Express Pilot Program

Federal Register 2010, 2011, 2012, 2013, 2014

2010-12-22

... SMALL BUSINESS ADMINISTRATION Community Express Pilot Program AGENCY: U.S. Small Business Administration (SBA). ACTION: Notice of short-term extension and termination of the Community Express Pilot Program. SUMMARY: This notice announces the termination of the Community Express Pilot Program following a...

Effects of different starch source of starter on small intestinal growth and endogenous GLP-2 secretion in preweaned lambs.

PubMed

Sun, Daming; Li, Hongwei; Mao, Shengyong; Zhu, Weiyun; Liu, Junhua

2018-02-15

The objective of this study was to investigate the effects of different sources of starch in starter feed on small intestinal growth and endogenous glucagon-like peptide 2 (GLP-2) secretion in preweaned lambs. Twenty-four 10-d-old lambs were divided into three groups that were treated with different iso-starch diets containing purified cassava starch (CS, n = 8), maize starch (MS, n = 8), and pea starch (PS, n = 8). At 56 d old, there was no significant difference in final body weight (BW) of lambs among the three groups. However, different starch source in starter significantly affected the average daily feed intake (ADFI) and average daily gain (ADG) of lambs among three groups. Compared with the CS and MS diets, the PS diet significantly increased the GLP-2 concentration in blood plasma (P < 0.001), the crypt depth of the jejunum (P = 0.006), and the villus height of the ileum (P = 0.039). Meanwhile, PS diet significantly increased the mRNA expression of proglucagon and the glucagon-like peptide 2 receptor (GLP-2R) in the jejunum and ileum (P < 0.001). Furthermore, the PS diet significantly upregulated the mRNA expression of cyclin D1 (P < 0.001), cyclin E (P = 0.006), and cyclin-dependent kinases 6 (CDK6) (P = 0.048) in the jejunum and cyclin A (P < 0.001), cyclin D1 (P < 0.001), and CDK6 (P = 0.002) in the ileum. Correlation analysis showed that endogenous GLP-2 secretion was positively related to the mRNA levels of cell cycle proteins in small intestinal mucosa. In summary, all results showed that PS in starter feed promoted small intestinal growth that may, in part, be related to cell cycle acceleration and endogenous GLP-2 secretion in preweaned lambs. These findings provide new insights into nutritional interventions that promote the development of small intestines in young ruminants.

Possible role of CD22, CD79b and CD20 expression in distinguishing small lymphocytic lymphoma from chronic lymphocytic leukemia.

PubMed

Jovanovic, Danijela; Djurdjevic, Predrag; Andjelkovic, Nebojsa; Zivic, Ljubica

2014-01-01

Flow cytometry has an important role in diagnosis and classification of B-cell lymphoproliferative disorders (BCLPDs). However, in distinguishing chronic lymphocytic leukemia (CLL) from small lymphocytic lymphoma (SLL) only clinical criteria are available so far. Aim of the study was to determine differences in the expression of common B cell markers (CD22, CD79b and CD20) on the malignant lymphocytes in the peripheral blood samples of CLL and SLL patients. Peripheral blood samples of 56 CLL and 11 SLL patients were analyzed by 5-color flow cytometry on the CD45/CD19/CD5 gate for CD22, CD79b and CD20. In the samples collected from the CLL patients, CD22 expression was detected in only 20% of patients in the low pattern, while in SLL patients the expression was medium and present in 90.9% of patients (p < 0.0001). For CD79b expression, statistical significance is reached both in the expression pattern, which was low/medium for CLL and high for SLL, and expression level (p = 0.006). The expression of CD20 was counted as the CD20/CD19 ratio. The average ratio was 0.512 in the CLL patients vs. 0.931 in the SLL patients (p = 0.0001). The pattern of expression and expression level of CD22, CD79b and CD20 in peripheral blood could be used for distinguishing SLL from CLL patients.

High expression of AID and active class switch recombination might account for a more aggressive disease in unmutated CLL patients: link with an activated microenvironment in CLL disease.

PubMed

Palacios, Florencia; Moreno, Pilar; Morande, Pablo; Abreu, Cecilia; Correa, Agustín; Porro, Valentina; Landoni, Ana Ines; Gabus, Raul; Giordano, Mirta; Dighiero, Guillermo; Pritsch, Otto; Oppezzo, Pablo

2010-06-03

Interaction of chronic lymphocytic leukemia (CLL) B cells with tissue microenvironment has been suggested to favor disease progression by promoting malignant B-cell growth. Previous work has shown expression in peripheral blood (PB) of CLL B cells of activation-induced cytidine deaminase (AID) among CLL patients with an unmutated (UM) profile of immunoglobulin genes and with ongoing class switch recombination (CSR) process. Because AID expression results from interaction with activated tissue microenvironment, we speculated whether the small subset with ongoing CSR is responsible for high levels of AID expression and could be derived from this particular microenvironment. In this work, we quantified AID expression and ongoing CSR in PB of 50 CLL patients and characterized the expression of different molecules related to microenvironment interaction. Our results show that among UM patients (1) high AID expression is restricted to the subpopulation of tumoral cells ongoing CSR; (2) this small subset expresses high levels of proliferation, antiapoptotic and progression markers (Ki-67, c-myc, Bcl-2, CD49d, and CCL3/4 chemokines). Overall, this work outlines the importance of a cellular subset in PB of UM CLL patients with a poor clinical outcome, high AID levels, and ongoing CSR, whose presence might be a hallmark of a recent contact with the microenvironment.

75 FR 473 - Community Express Pilot Program

Federal Register 2010, 2011, 2012, 2013, 2014

2010-01-05

... SMALL BUSINESS ADMINISTRATION Community Express Pilot Program AGENCY: U.S. Small Business Administration (SBA). ACTION: Notice of extension of the Community Express Pilot Program. SUMMARY: This notice extends the Community Express Pilot Program in its current form through December 31, 2010. Based upon the...

Reputation and Parental Logics of Action in Local School Choice Space in Finland

ERIC Educational Resources Information Center

Kosunen, Sonja

2014-01-01

Differences in reputation between schools and in classes within schools shape parental choice in the Finnish urban context, even if the differences in school performance and the risks of making a "bad" choice are relatively small. This study analyses the instrumental and expressive orders of schools in a specific educational context. Two…

Analysis of dual coupler nested coupled cavities.

PubMed

Adib, George A; Sabry, Yasser M; Khalil, Diaa

2017-12-01

Coupled ring resonators are now forming the basic building blocks in several optical systems serving different applications. In many of these applications, a small full width at half maximum is required, along with a large free spectral range. In this work, a configuration of passive coupled cavities constituting dual coupler nested cavities is proposed. A theoretical study of the configuration is presented allowing us to obtain analytical expressions of its different spectral characteristics. The transfer function of the configuration is also used to generate design curves while comparing these results with analytical expressions. Finally, the configuration is compared with other coupled cavity configurations.

Enhanced expression and purification of camelid single domain VHH antibodies from classical inclusion bodies.

PubMed

Maggi, Maristella; Scotti, Claudia

2017-08-01

Single domain antibodies (sdAbs) are small antigen-binding domains derived from naturally occurring, heavy chain-only immunoglobulins isolated from camelid and sharks. They maintain the same binding capability of full-length IgGs but with improved thermal stability and permeability, which justifies their scientific, medical and industrial interest. Several described recombinant forms of sdAbs have been produced in different hosts and with different strategies. Here we present an optimized method for a time-saving, high yield production and extraction of a poly-histidine-tagged sdAb from Escherichia coli classical inclusion bodies. Protein expression and extraction were attempted using 4 different methods (e.g. autoinducing or IPTG-induced soluble expression, non-classical and classical inclusion bodies). The best method resulted to be expression in classical inclusion bodies and urea-mediated protein extraction which yielded 60-70 mg/l bacterial culture. The method we here describe can be of general interest for an enhanced and efficient heterologous expression of sdAbs for research and industrial purposes. Copyright © 2017 Elsevier Inc. All rights reserved.

Expression of fragile X mental retardation protein and Fmr1 mRNA during folliculogenesis in the rat.

PubMed

Ferder, Ianina; Parborell, Fernanda; Sundblad, Victoria; Chiauzzi, Violeta; Gómez, Karina; Charreau, Eduardo H; Tesone, Marta; Dain, Liliana

2013-04-01

Fragile X mental retardation protein (FMRP) belongs to a small family of RNA-binding proteins. Its absence or inactivity is responsible for fragile X syndrome, the most common cause of inherited mental retardation. Despite its ubiquitous expression, FMRP function and expression remain almost understudied in non-neuronal tissues, though previous studies on germline development during oogenesis may suggest a special function of this protein also in ovarian tissue. In addition, the well-documented association of FMR1 premutation state with fragile X-related premature ovarian insufficiency adds interest to the role of FMRP in ovarian physiology. The aim of the present work was to investigate the expression of Fmr1 mRNA and its protein, FMRP, at different stages of rat follicular development. By immunohistochemical studies we demonstrated FMRP expression in granulosa, theca and germ cells in all stages of follicular development. In addition, changes in Fmr1 expression, both at the protein and mRNA levels, were observed. FMRP levels increased upon follicular development while preantral and early antral follicles presented similar levels of Fmr1 transcripts with decreased expression in preovulatory follicles. These observations suggest that Fmr1 expression in the ovary is regulated at different and perhaps independent levels. In addition, our results show expression of at least four different isoforms of FMRP during all stages of follicular growth with expression patterns that differ from those observed in brain and testis. Our study shows a regulated expression of Fmr1, both at mRNA and protein levels, during rat follicular development.

Functional and structural analysis of a highly-expressed Yersinia pestis small RNA following infection of cultured macrophages

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Nan; Hennelly, Scott P.; Stubben, Chris J.

Non-coding small RNAs (sRNAs) are found in practically all bacterial genomes and play important roles in regulating gene expression to impact bacterial metabolism, growth, and virulence. We performed transcriptomics analysis to identify sRNAs that are differentially expressed in Yersinia pestis that invaded the human macrophage cell line THP-1, compared to pathogens that remained extracellular in the presence of host. Using ultra high-throughput sequencing, we identified 37 novel and 143 previously known sRNAs in Y. pestis. In particular, the sRNA Ysr170 was highly expressed in intracellular Yersinia and exhibited a log2 fold change ~3.6 higher levels compared to extracellular bacteria. Wemore » found that knock-down of Ysr170 expression attenuated infection efficiency in cell culture and growth rate in response to different stressors. In addition, we applied selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE) analysis to determine the secondary structure of Ysr170 and observed structural changes resulting from interactions with the aminoglycoside antibiotic gentamycin and the RNA chaperone Hfq. Interestingly, gentamicin stabilized helix 4 of Ysr170, which structurally resembles the native gentamicin 16S ribosomal binding site. Lastly, we modeled the tertiary structure of Ysr170 binding to gentamycin using RNA motif modeling. Integration of these experimental and structural methods can provide further insight into the design of small molecules that can inhibit function of sRNAs required for pathogen virulence.« less

Functional and structural analysis of a highly-expressed Yersinia pestis small RNA following infection of cultured macrophages

DOE PAGES

Li, Nan; Hennelly, Scott P.; Stubben, Chris J.; ...

2016-12-28

Non-coding small RNAs (sRNAs) are found in practically all bacterial genomes and play important roles in regulating gene expression to impact bacterial metabolism, growth, and virulence. We performed transcriptomics analysis to identify sRNAs that are differentially expressed in Yersinia pestis that invaded the human macrophage cell line THP-1, compared to pathogens that remained extracellular in the presence of host. Using ultra high-throughput sequencing, we identified 37 novel and 143 previously known sRNAs in Y. pestis. In particular, the sRNA Ysr170 was highly expressed in intracellular Yersinia and exhibited a log2 fold change ~3.6 higher levels compared to extracellular bacteria. Wemore » found that knock-down of Ysr170 expression attenuated infection efficiency in cell culture and growth rate in response to different stressors. In addition, we applied selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE) analysis to determine the secondary structure of Ysr170 and observed structural changes resulting from interactions with the aminoglycoside antibiotic gentamycin and the RNA chaperone Hfq. Interestingly, gentamicin stabilized helix 4 of Ysr170, which structurally resembles the native gentamicin 16S ribosomal binding site. Lastly, we modeled the tertiary structure of Ysr170 binding to gentamycin using RNA motif modeling. Integration of these experimental and structural methods can provide further insight into the design of small molecules that can inhibit function of sRNAs required for pathogen virulence.« less

75 FR 77935 - Patriot Express Pilot Loan Initiative

Federal Register 2010, 2011, 2012, 2013, 2014

2010-12-14

... SMALL BUSINESS ADMINISTRATION Patriot Express Pilot Loan Initiative AGENCY: U.S. Small Business Administration (SBA). ACTION: Notice of extension of the Patriot Express Pilot Loan Initiative. SUMMARY: This notice extends the Patriot Express Pilot Loan Initiative in its current form through December 31, 2013...

The miiuy croaker microRNA transcriptome and microRNA regulation of RIG-I like receptor signaling pathway after poly(I:C) stimulation.

PubMed

Han, Jingjing; Xu, Guoliang; Xu, Tianjun

2016-07-01

MicroRNAs (miRNAs) as endogenous small non-coding RNAs play key regulatory roles in diverse biological processes via degrading the target mRNAs or inhibiting protein translation. Previously many researchers have reported the identification, characteristic of miRNAs and the interaction with its target gene. But, the study on the regulation of miRNAs to biological processes via regulatory the key signaling pathway was still limited. In order to comprehend the regulatory mechanism of miRNAs, two small RNA libraries from the spleen of miiuy croaker individuals with or without poly(I:C) infection were constructed. The 197 conserved miRNAs and 75 novel miRNAs were identified, and 14 conserved and 8 novel miRNAs appeared significant variations. Those differently expressed miRNAs relate to immune regulation of miiuy croaker. Furthermore, expressions of four differently expressed miRNAs were validated by qRT-PCR, and the result was consistent with sequencing data. The target genes of the differently expressed miRNAs in the two libraries were predicted, and some candidate target genes were involved in the RIG-I-like receptor (RLR) signaling pathway. The negative regulation of miRNAs to target genes were confirmed by comparing the expression pattern of miRNAs and their target genes. The results of regulating target genes were that firstly directly or indirectly activating the downstream signaling cascades and subsequent inducting the type I interferon, inflammatory cytokines and apoptosis. These studies could help us to deeper understand the roles of miRNAs played in the fish immune system, and provide a new way to investigate the defense mechanism of fish. Copyright © 2016 Elsevier Ltd. All rights reserved.

Employing conservation of co-expression to improve functional inference

PubMed Central

Daub, Carsten O; Sonnhammer, Erik LL

2008-01-01

Background Observing co-expression between genes suggests that they are functionally coupled. Co-expression of orthologous gene pairs across species may improve function prediction beyond the level achieved in a single species. Results We used orthology between genes of the three different species S. cerevisiae, D. melanogaster, and C. elegans to combine co-expression across two species at a time. This led to increased function prediction accuracy when we incorporated expression data from either of the other two species and even further increased when conservation across both of the two other species was considered at the same time. Employing the conservation across species to incorporate abundant model organism data for the prediction of protein interactions in poorly characterized species constitutes a very powerful annotation method. Conclusion To be able to employ the most suitable co-expression distance measure for our analysis, we evaluated the ability of four popular gene co-expression distance measures to detect biologically relevant interactions between pairs of genes. For the expression datasets employed in our co-expression conservation analysis above, we used the GO and the KEGG PATHWAY databases as gold standards. While the differences between distance measures were small, Spearman correlation showed to give most robust results. PMID:18808668

Small Heat Shock Proteins Can Release Light Dependence of Tobacco Seed during Germination1[OPEN

PubMed Central

Koo, Hyun Jo; Park, Soo Min; Kim, Keun Pill; Suh, Mi Chung; Lee, Mi Ok; Lee, Seong-Kon; Xinli, Xia

2015-01-01

Small heat shock proteins (sHSPs) function as ATP-independent molecular chaperones, and although the production and function of sHSPs have often been described under heat stress, the expression and function of sHSPs in fundamental developmental processes, such as pollen and seed development, have also been confirmed. Seed germination involves the breaking of dormancy and the resumption of embryo growth that accompany global changes in transcription, translation, and metabolism. In many plants, germination is triggered simply by imbibition of water; however, different seeds require different conditions in addition to water. For small-seeded plants, like Arabidopsis (Arabidopsis thaliana), lettuce (Lactuca sativa), tomato (Solanum lycopersicum), and tobacco (Nicotiana tabacum), light is an important regulator of seed germination. The facts that sHSPs accumulate during seed development, sHSPs interact with various client proteins, and seed germination accompanies synthesis and/or activation of diverse proteins led us to investigate the role of sHSPs in seed germination, especially in the context of light dependence. In this study, we have built transgenic tobacco plants that ectopically express sHSP, and the effect was germination of the seeds in the dark. Administering heat shock to the seeds also resulted in the alleviation of light dependence during seed germination. Subcellular localization of ectopically expressed sHSP was mainly observed in the cytoplasm, whereas heat shock-induced sHSPs were transported to the nucleus. We hypothesize that ectopically expressed sHSPs in the cytoplasm led the status of cytoplasmic proteins involved in seed germination to function during germination without additional stimulus and that heat shock can be another signal that induces seed germination. PMID:25604531

Long interspersed nuclear elements (LINEs) show tissue-specific, mosaic genome and methylation-unrestricted, widespread expression of noncoding RNAs in somatic tissues of the rat

PubMed Central

Singh, Deepak K.; Rath, Pramod C.

2012-01-01

We report strong somatic and germ line expression of LINE RNAs in eight different tissues of rat by using a novel ~2.8 kb genomic PstI-LINE DNA (P1-LINE) isolated from the rat brain. P1-LINE is present in a 93 kb LINE-SINE-cluster in sub-telomeric region of chromosome 12 (12p12) and as multiple truncated copies interspersed in all rat chromosomes. P1-LINEs occur as inverted repeats at multiple genomic loci in tissue-specific and mosaic patterns. P1-LINE RNAs are strongly expressed in brain, liver, lungs, heart, kidney, testes, spleen and thymus into large to small heterogeneous RNAs (~5.0 to 0.2 kb) in tissue-specific and dynamic patterns in individual rats. P1-LINE DNA is strongly methylated at CpG-dinucleotides in most genomic copies in all the tissues and weakly hypomethylated in few copies in some tissues. Small (700–75 nt) P1-LINE RNAs expressed in all tissues may be possible precursors for small regulatory RNAs (PIWI-interacting/piRNAs) bioinformatically derived from P1-LINE. The strong and dynamic expression of LINE RNAs from multiple chromosomal loci and the putative piRNAs in somatic tissues of rat under normal physiological conditions may define functional chromosomal domains marked by LINE RNAs as long noncoding RNAs (lncRNAs) unrestricted by DNA methylation. The tissue-specific, dynamic RNA expression and mosaic genomic distribution of LINEs representing a steady-state genomic flux of retrotransposon RNAs suggest for biological role of LINE RNAs as long ncRNAs and small piRNAs in mammalian tissues independent of their cellular fate for translation, reverse-transcription and retrotransposition. This may provide evolutionary advantages to LINEs and mammalian genomes. PMID:23064113

Ovarian stem cells are always accompanied by very small embryonic-like stem cells in adult mammalian ovary.

PubMed

Bhartiya, Deepa

2015-11-05

Existing dogma that a female is born with fixed number of eggs was challenged by the detection of stem cells in adult mammalian ovary. Data has accumulated in support of ovarian stem cells (OSCs) proliferation, maintenance in culture, formation of germ cell nests and differentiation into oocytes and primordial follicle assembly using different strategies. Flow cytometry analysis identified >8 μm OSCs which are DDX1 positive and are considered equivalent to spermatogonial stem cells (SSCs) in testis. Analysis of both ovarian and testicular smears obtained after enzymatic digestion has led to the identification of an additional stem cell population termed very small embryonic-like stem cells (VSELs). VSELs and OSCs/SSCs differ from each other in their size and OCT-4 expression. VSELs express pluripotent markers including nuclear OCT-4 whereas OSCs/SSCs express cytoplasmic OCT-4 suggesting a differentiated state. VSELs can be studied by flow cytometry as small sized cells which are LIN-/CD45-/Sca-1+. We have reported 0.02 ± 0.008, 0.03 ± 0.017 and 0.08 ± 0.03 % of total cells as VSELs in normal, chemoablated and after FSH treatment to chemoablated mouse ovary. VSELs have remained poorly studied till now because of their very small size and rare occurrence. Spinning cells obtained after enzymatic digestion of ovarian tissue at a speed of 1000G (rather than 1200 rpm) throughout processing allows reliable detection of the VSELs by flow cytometry. VSELs exist in aged, chemoablated and non-functional ovary and providing a healthy niche to support their function offers an interesting strategy to manage infertility.

CCI-007, a novel small molecule with cytotoxic activity against infant leukemia with MLL rearrangements

PubMed Central

Middlemiss, Shiloh M.C.; Wen, Victoria W.; Clifton, Molly; Kwek, Alan; Liu, Bing; Mayoh, Chelsea; Bongers, Angelika; Karsa, Mawar; Pan, Sukey; Cruikshank, Sarah; Scandlyn, Marissa; Hoang, Wendi; Imamura, Toshihiko; Kees, Ursula R.; Gudkov, Andrei V.; Chernova, Olga B.

2016-01-01

There is an urgent need for the development of less toxic, more selective and targeted therapies for infants with leukemia characterized by translocation of the mixed lineage leukemia (MLL) gene. In this study, we performed a cell-based small molecule library screen on an infant MLL-rearranged (MLL-r) cell line, PER-485, in order to identify selective inhibitors for MLL-r leukemia. After screening initial hits for a cytotoxic effect against a panel of 30 cell lines including MLL-r and MLL wild-type (MLL-wt) leukemia, solid tumours and control cells, small molecule CCI-007 was identified as a compound that selectively and significantly decreased the viability of a subset of MLL-r and related leukemia cell lines with CALM-AF10 and SET-NUP214 translocation. CCI-007 induced a rapid caspase-dependent apoptosis with mitochondrial depolarization within twenty-four hours of treatment. CCI-007 altered the characteristic MLL-r gene expression signature in sensitive cells with downregulation of the expression of HOXA9, MEIS1, CMYC and BCL2, important drivers in MLL-r leukemia, within a few hours of treatment. MLL-r leukemia cells that were resistant to the compound were characterised by significantly higher baseline gene expression levels of MEIS1 and BCL2 in comparison to CCI-007 sensitive MLL-r leukemia cells. In conclusion, we have identified CCI-007 as a novel small molecule that displays rapid toxicity towards a subset of MLL-r, CALM-AF10 and SET-NUP214 leukemia cell lines. Our findings suggest an important new avenue in the development of targeted therapies for these deadly diseases and indicate that different therapeutic strategies might be needed for different subtypes of MLL-r leukemia. PMID:27317766

Intermediate filament protein nestin is expressed in developing meninges.

PubMed

Yay, A; Ozdamar, S; Canoz, O; Baran, M; Tucer, B; Sonmez, M F

2014-01-01

Nestin is a type VI intermediate filament protein known as a marker for progenitor cells that can be mostly found in tissues during the embryonic and fetal periods. In our study, we aimed to determine the expression of nestin in meninges covering the brain tissue at different developmental stages and in the new born. In this study 10 human fetuses in different development stages between developmental weeks 9-34 and a newborn brain tissue were used. Fetuses in paraffin section were stained with H+E and nestin immunohistochemical staining protocol was performed. In this study, in the human meninges intense nestin expression was detected as early as in the 9th week of development. Intensity of this expression gradually decreased in later stages of development and nestin expression still persisted in a small population of newborn meningeal cells. In the present study, nestin positive cells gradually diminished in the developing and maturing meninges during the fetal period. This probably depends on initiation of a decrease in nestin expression and replacement with other tissue-specific intermediate filaments while the differentiation process continues. These differences can make significant contributions to the investigation and diagnosis of various pathological disorders (Tab. 1, Fig. 3, Ref. 36).

Generation of 2A-linked multicistronic cassettes by recombinant PCR.

PubMed

Szymczak-Workman, Andrea L; Vignali, Kate M; Vignali, Dario A A

2012-02-01

The need for reliable, multicistronic vectors for multigene delivery is at the forefront of biomedical technology. It is now possible to express multiple proteins from a single open reading frame (ORF) using 2A peptide-linked multicistronic vectors. These small sequences, when cloned between genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel "cleavage" event within the 2A peptide sequence. Expression of more than two genes using conventional approaches has several limitations, most notably imbalanced protein expression and large size. The use of 2A peptide sequences alleviates these concerns. They are small (18-22 amino acids) and have divergent amino-terminal sequences, which minimizes the chance for homologous recombination and allows for multiple, different 2A peptide sequences to be used within a single vector. Importantly, separation of genes placed between 2A peptide sequences is nearly 100%, which allows for stoichiometric and concordant expression of the genes, regardless of the order of placement within the vector. This protocol describes the use of recombinant polymerase chain reaction (PCR) to connect multiple 2A-linked protein sequences. The final construct is subcloned into an expression vector.

Different sucrose-isomaltase response of Caco-2 cells to glucose and maltose suggests dietary maltose sensing

PubMed Central

Cheng, Min-Wen; Chegeni, Mohammad; Kim, Kee-Hong; Zhang, Genyi; Benmoussa, Mustapha; Quezada-Calvillo, Roberto; Nichols, Buford L.; Hamaker, Bruce R.

2014-01-01

Using the small intestine enterocyte Caco-2 cell model, sucrase-isomaltase (SI, the mucosal α-glucosidase complex) expression and modification were examined relative to exposure to different mono- and disaccharide glycemic carbohydrates. Caco-2/TC7 cells were grown on porous supports to post-confluence for complete differentiation, and dietary carbohydrate molecules of glucose, sucrose (disaccharide of glucose and fructose), maltose (disaccharide of two glucoses α-1,4 linked), and isomaltose (disaccharide of two glucoses α-1,6 linked) were used to treat the cells. qRT-PCR results showed that all the carbohydrate molecules induced the expression of the SI gene, though maltose (and isomaltose) showed an incremental increase in mRNA levels over time that glucose did not. Western blot analysis of the SI protein revealed that only maltose treatment induced a higher molecular weight band (Mw ~245 kDa), also at higher expression level, suggesting post-translational processing of SI, and more importantly a sensing of maltose. Further work is warranted regarding this putative sensing response as a potential control point for starch digestion and glucose generation in the small intestine. PMID:24426192

Different sucrose-isomaltase response of Caco-2 cells to glucose and maltose suggests dietary maltose sensing.

PubMed

Cheng, Min-Wen; Chegeni, Mohammad; Kim, Kee-Hong; Zhang, Genyi; Benmoussa, Mustapha; Quezada-Calvillo, Roberto; Nichols, Buford L; Hamaker, Bruce R

2014-01-01

Using the small intestine enterocyte Caco-2 cell model, sucrase-isomaltase (SI, the mucosal α-glucosidase complex) expression and modification were examined relative to exposure to different mono- and disaccharide glycemic carbohydrates. Caco-2/TC7 cells were grown on porous supports to post-confluence for complete differentiation, and dietary carbohydrate molecules of glucose, sucrose (disaccharide of glucose and fructose), maltose (disaccharide of two glucoses α-1,4 linked), and isomaltose (disaccharide of two glucoses α-1,6 linked) were used to treat the cells. qRT-PCR results showed that all the carbohydrate molecules induced the expression of the SI gene, though maltose (and isomaltose) showed an incremental increase in mRNA levels over time that glucose did not. Western blot analysis of the SI protein revealed that only maltose treatment induced a higher molecular weight band (Mw ~245 kDa), also at higher expression level, suggesting post-translational processing of SI, and more importantly a sensing of maltose. Further work is warranted regarding this putative sensing response as a potential control point for starch digestion and glucose generation in the small intestine.

Apparently low reproducibility of true differential expression discoveries in microarray studies.

PubMed

Zhang, Min; Yao, Chen; Guo, Zheng; Zou, Jinfeng; Zhang, Lin; Xiao, Hui; Wang, Dong; Yang, Da; Gong, Xue; Zhu, Jing; Li, Yanhui; Li, Xia

2008-09-15

Differentially expressed gene (DEG) lists detected from different microarray studies for a same disease are often highly inconsistent. Even in technical replicate tests using identical samples, DEG detection still shows very low reproducibility. It is often believed that current small microarray studies will largely introduce false discoveries. Based on a statistical model, we show that even in technical replicate tests using identical samples, it is highly likely that the selected DEG lists will be very inconsistent in the presence of small measurement variations. Therefore, the apparently low reproducibility of DEG detection from current technical replicate tests does not indicate low quality of microarray technology. We also demonstrate that heterogeneous biological variations existing in real cancer data will further reduce the overall reproducibility of DEG detection. Nevertheless, in small subsamples from both simulated and real data, the actual false discovery rate (FDR) for each DEG list tends to be low, suggesting that each separately determined list may comprise mostly true DEGs. Rather than simply counting the overlaps of the discovery lists from different studies for a complex disease, novel metrics are needed for evaluating the reproducibility of discoveries characterized with correlated molecular changes. Supplementaty information: Supplementary data are available at Bioinformatics online.

CXCR6 expression in non-small cell lung carcinoma supports metastatic process via modulating metalloproteinases.

PubMed

Mir, Hina; Singh, Rajesh; Kloecker, Goetz H; Lillard, James W; Singh, Shailesh

2015-04-30

Lung cancer (LuCa) is the leading cause of cancer-related deaths worldwide regardless of the gender. High mortality associated with LuCa is due to metastasis, molecular mechanisms of which are yet to be defined. Here, we present evidence that chemokine receptor CXCR6 and its only natural ligand, CXCL16, are significantly expressed by non-small cell lung cancer (NSCLC) and are involved in the pathobiology of LuCa. CXCR6 expression was significantly higher in two subtypes of NSCLC (adenocarcinomas-ACs and squamous cell carcinoma-SCCs) as compared to non-neoplastic tissue. Additionally, serum CXCL16 was significantly elevated in LuCa cases as compared to healthy controls. Similar to CXCR6 tissue expression, serum level of CXCL16 in AC patients was significantly higher than SCC patients. Biological significance of this axis was validated using SCC and AC cell lines. Expression of CXCR6 was higher in AC cells, which also showed higher migratory and invasive potential than SCC. Differences in migratory and invasive potential between AC and SCC were due to differential expression of metalloproteinases following CXCL16 stimulation. Hence, our findings suggest clinical and biological significance of CXCR6/CXCL16 axis in LuCa, which could be used as potential prognostic marker and therapeutic target.

CXCR6 expression in non-small cell lung carcinoma supports metastatic process via modulating metalloproteinases

PubMed Central

Mir, Hina; Singh, Rajesh; Kloecker, Goetz H.; Lillard, James W.; Singh, Shailesh

2015-01-01

Lung cancer (LuCa) is the leading cause of cancer-related deaths worldwide regardless of the gender. High mortality associated with LuCa is due to metastasis, molecular mechanisms of which are yet to be defined. Here, we present evidence that chemokine receptor CXCR6 and its only natural ligand, CXCL16, are significantly expressed by non-small cell lung cancer (NSCLC) and are involved in the pathobiology of LuCa. CXCR6 expression was significantly higher in two subtypes of NSCLC (adenocarcinomas-ACs and squamous cell carcinoma-SCCs) as compared to non-neoplastic tissue. Additionally, serum CXCL16 was significantly elevated in LuCa cases as compared to healthy controls. Similar to CXCR6 tissue expression, serum level of CXCL16 in AC patients was significantly higher than SCC patients. Biological significance of this axis was validated using SCC and AC cell lines. Expression of CXCR6 was higher in AC cells, which also showed higher migratory and invasive potential than SCC. Differences in migratory and invasive potential between AC and SCC were due to differential expression of metalloproteinases following CXCL16 stimulation. Hence, our findings suggest clinical and biological significance of CXCR6/CXCL16 axis in LuCa, which could be used as potential prognostic marker and therapeutic target. PMID:25888629

GC-rich coding sequences reduce transposon-like, small RNA-mediated transgene silencing.

PubMed

Sidorenko, Lyudmila V; Lee, Tzuu-Fen; Woosley, Aaron; Moskal, William A; Bevan, Scott A; Merlo, P Ann Owens; Walsh, Terence A; Wang, Xiujuan; Weaver, Staci; Glancy, Todd P; Wang, PoHao; Yang, Xiaozeng; Sriram, Shreedharan; Meyers, Blake C

2017-11-01

The molecular basis of transgene susceptibility to silencing is poorly characterized in plants; thus, we evaluated several transgene design parameters as means to reduce heritable transgene silencing. Analyses of Arabidopsis plants with transgenes encoding a microalgal polyunsaturated fatty acid (PUFA) synthase revealed that small RNA (sRNA)-mediated silencing, combined with the use of repetitive regulatory elements, led to aggressive transposon-like silencing of canola-biased PUFA synthase transgenes. Diversifying regulatory sequences and using native microalgal coding sequences (CDSs) with higher GC content improved transgene expression and resulted in a remarkable trans-generational stability via reduced accumulation of sRNAs and DNA methylation. Further experiments in maize with transgenes individually expressing three crystal (Cry) proteins from Bacillus thuringiensis (Bt) tested the impact of CDS recoding using different codon bias tables. Transgenes with higher GC content exhibited increased transcript and protein accumulation. These results demonstrate that the sequence composition of transgene CDSs can directly impact silencing, providing design strategies for increasing transgene expression levels and reducing risks of heritable loss of transgene expression.

Design and construction of 2A peptide-linked multicistronic vectors.

PubMed

Szymczak-Workman, Andrea L; Vignali, Kate M; Vignali, Dario A A

2012-02-01

The need for reliable, multicistronic vectors for multigene delivery is at the forefront of biomedical technology. This article describes the design and construction of 2A peptide-linked multicistronic vectors, which can be used to express multiple proteins from a single open reading frame (ORF). The small 2A peptide sequences, when cloned between genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel "cleavage" event within the 2A peptide sequence. Expression of more than two genes using conventional approaches has several limitations, most notably imbalanced protein expression and large size. The use of 2A peptide sequences alleviates these concerns. They are small (18-22 amino acids) and have divergent amino-terminal sequences, which minimizes the chance for homologous recombination and allows for multiple, different 2A peptide sequences to be used within a single vector. Importantly, separation of genes placed between 2A peptide sequences is nearly 100%, which allows for stoichiometric and concordant expression of the genes, regardless of the order of placement within the vector.

omiRas: a Web server for differential expression analysis of miRNAs derived from small RNA-Seq data.

PubMed

Müller, Sören; Rycak, Lukas; Winter, Peter; Kahl, Günter; Koch, Ina; Rotter, Björn

2013-10-15

Small RNA deep sequencing is widely used to characterize non-coding RNAs (ncRNAs) differentially expressed between two conditions, e.g. healthy and diseased individuals and to reveal insights into molecular mechanisms underlying condition-specific phenotypic traits. The ncRNAome is composed of a multitude of RNAs, such as transfer RNA, small nucleolar RNA and microRNA (miRNA), to name few. Here we present omiRas, a Web server for the annotation, comparison and visualization of interaction networks of ncRNAs derived from next-generation sequencing experiments of two different conditions. The Web tool allows the user to submit raw sequencing data and results are presented as: (i) static annotation results including length distribution, mapping statistics, alignments and quantification tables for each library as well as lists of differentially expressed ncRNAs between conditions and (ii) an interactive network visualization of user-selected miRNAs and their target genes based on the combination of several miRNA-mRNA interaction databases. The omiRas Web server is implemented in Python, PostgreSQL, R and can be accessed at: http://tools.genxpro.net/omiras/.

[Tripartite-motif protein 25 and pyruvate kinase M2 protein expression in non-small cell lung cancer].

PubMed

Jing, Huai-Zhi; Qiu, Feng; Chen, Shi-Zhi; Su, Lin; Qu, Can

2015-03-01

To investigate the expression of tripartite-motif protein 25 (TRIM25) and pyruvate kinase M2 (PKM2) protein in non-small cell lung cancer (NSCLC) and explore their role in the occurrence and progression of NSCLC. The expressions of TRIM25 and PKM2 protein were detected in 60 NSCLC specimens and 20 adjacent normal lung tissue (>5 cm from the lesions) with immunofluorescence histochemical method and in 10 fresh specimens of NSCLC with Western blotting. The results were analyzed in relation with the clinicopathological features of the patients. The positivity rates of TRIM25 expression was 45% in the 60 lung carcinoma specimens, significantly higher than that in the 20 normal lung tissues (10%, P=0.005). TRIM25 protein was expressed in 28.6% of lung adenocarcinoma tissues and in 59.4% of squamous carcinoma tissues (P=0.017). TRIM25 protein expression was positively correlated with the TNM stages and lymph node metastasis of NSCLC (P<0.05). The expressions of PKM2 protein in 60 cases of lung carcinoma was 73.3%,while in 20 cases of normal lung tissues the expressions was 30%(P=0.001). The positivity rates of PKM2 expression differed significantly between lung adenocarcinoma and squamous carcinoma (57.1% vs 87.5%, P=0.008). An inverse correlation was noted between TRIM25 and PKM2 expressions (P=0.026). TRIM25 and PKM2 protein may participate in the occurrence and progression of NSCLC, and their expressions are inversely correlated.

High-throughput deep screening and identification of four peripheral leucocyte microRNAs as novel potential combination biomarkers for preeclampsia

PubMed Central

Wang, Yonghong; Yang, Xukui; Yang, Yuanyuan; Wang, Wenjun; Zhao, Meiling; Liu, Huiqiang; Li, Dongyan; Hao, Min

2016-01-01

Objective: To identify the specific microRNA (miRNA) biomarkers of preeclampsia (PE), the miRNA profiles analysis were performed. Study Design: The blood samples were obtained from five PE patients and five normal healthy pregnant women. The small RNA profiles were analyzed to identify miRNA expression levels and find out miRNAs that may associate with PE. The quantitative reverse transcriptase–PCR (qRT-PCR) assay was used to validate differentially expressed peripheral leucocyte miRNAs in a new cohort. Result: The data analysis showed that 10 peripheral leucocyte miRNAs were significantly differently expressed in severe PE patients. Four differently expressed miRNAs were successfully validated using qRT-PCR method. Conclusion: We successfully constructed a model with high accuracy to predict PE. A combination of four peripheral leucocyte miRNAs has great potential to serve as diagnostic biomarkers of PE. PMID:26675000

Small Molecule Supplements Improve Cultured Megakaryocyte Polyploidization by Modulating Multiple Cell Cycle Regulators.

PubMed

Zou, Xiaojing; Qu, Mingyi; Fang, Fang; Fan, Zeng; Chen, Lin; Yue, Wen; Xie, Xiaoyan; Pei, Xuetao

2017-01-01

Platelets (PLTs) are produced by megakaryocytes (MKs) that completed differentiation and endomitosis. Endomitosis is an important process in which the cell replicates its DNA without cytokinesis and develops highly polyploid MK. In this study, to gain a better PLTs production, four small molecules (Rho-Rock inhibitor (RRI), nicotinamide (NIC), Src inhibitor (SI), and Aurora B inhibitor (ABI)) and their combinations were surveyed as MK culture supplements for promoting polyploidization. Three leukemia cell lines as well as primary mononuclear cells were chosen in the function and mechanism studies of the small molecules. In an optimal culture method, cells were treated with different small molecules and their combinations. The impact of the small molecules on megakaryocytic surface marker expression, polyploidy, proliferation, and apoptosis was examined for the best MK polyploidization supplement. The elaborate analysis confirmed that the combination of SI and RRI together with our MK induction system might result in efficient ploidy promotion. Our experiments demonstrated that, besides direct downregulation on the expression of cytoskeleton protein actin, SI and RRI could significantly enhance the level of cyclins through the suppression of p53 and p21. The verified small molecule combination might be further used in the in vitro PLT manufacture and clinical applications.

Small Molecule Supplements Improve Cultured Megakaryocyte Polyploidization by Modulating Multiple Cell Cycle Regulators

PubMed Central

Fang, Fang; Chen, Lin; Yue, Wen

2017-01-01

Platelets (PLTs) are produced by megakaryocytes (MKs) that completed differentiation and endomitosis. Endomitosis is an important process in which the cell replicates its DNA without cytokinesis and develops highly polyploid MK. In this study, to gain a better PLTs production, four small molecules (Rho-Rock inhibitor (RRI), nicotinamide (NIC), Src inhibitor (SI), and Aurora B inhibitor (ABI)) and their combinations were surveyed as MK culture supplements for promoting polyploidization. Three leukemia cell lines as well as primary mononuclear cells were chosen in the function and mechanism studies of the small molecules. In an optimal culture method, cells were treated with different small molecules and their combinations. The impact of the small molecules on megakaryocytic surface marker expression, polyploidy, proliferation, and apoptosis was examined for the best MK polyploidization supplement. The elaborate analysis confirmed that the combination of SI and RRI together with our MK induction system might result in efficient ploidy promotion. Our experiments demonstrated that, besides direct downregulation on the expression of cytoskeleton protein actin, SI and RRI could significantly enhance the level of cyclins through the suppression of p53 and p21. The verified small molecule combination might be further used in the in vitro PLT manufacture and clinical applications. PMID:29201898

A Unique cis-Encoded Small Noncoding RNA Is Regulating Legionella pneumophila Hfq Expression in a Life Cycle-Dependent Manner.

PubMed

Oliva, Giulia; Sahr, Tobias; Rolando, Monica; Knoth, Maike; Buchrieser, Carmen

2017-01-10

Legionella pneumophila is an environmental bacterium that parasitizes protozoa, but it may also infect humans, thereby causing a severe pneumonia called Legionnaires' disease. To cycle between the environment and a eukaryotic host, L. pneumophila is regulating the expression of virulence factors in a life cycle-dependent manner: replicating bacteria do not express virulence factors, whereas transmissive bacteria are highly motile and infective. Here we show that Hfq is an important regulator in this network. Hfq is highly expressed in transmissive bacteria but is expressed at very low levels in replicating bacteria. A L. pneumophila hfq deletion mutant exhibits reduced abilities to infect and multiply in Acanthamoeba castellanii at environmental temperatures. The life cycle-dependent regulation of Hfq expression depends on a unique cis-encoded small RNA named Anti-hfq that is transcribed antisense of the hfq transcript and overlaps its 5' untranslated region. The Anti-hfq sRNA is highly expressed only in replicating L. pneumophila where it regulates hfq expression through binding to the complementary regions of the hfq transcripts. This results in reduced Hfq protein levels in exponentially growing cells. Both the small noncoding RNA (sRNA) and hfq mRNA are bound and stabilized by the Hfq protein, likely leading to the cleavage of the RNA duplex by the endoribonuclease RNase III. In contrast, after the switch to transmissive bacteria, the sRNA is not expressed, allowing now an efficient expression of the hfq gene and consequently Hfq. Our results place Hfq and its newly identified sRNA anti-hfq in the center of the regulatory network governing L. pneumophila differentiation from nonvirulent to virulent bacteria. The abilities of L. pneumophila to replicate intracellularly and to cause disease depend on its capacity to adapt to different extra- and intracellular environmental conditions. Therefore, a timely and fine-tuned expression of virulence factors and adaptation traits is crucial. Yet, the regulatory circuits governing the life cycle of L. pneumophila from replicating to virulent bacteria are only partly uncovered. Here we show that the life cycle-dependent regulation of the RNA chaperone Hfq relies on a small regulatory RNA encoded antisense to the hfq-encoding gene through a base pairing mechanism. Furthermore, Hfq regulates its own expression in an autoregulatory loop. The discovery of this RNA regulatory mechanism in L. pneumophila is an important step forward in the understanding of how the switch from inoffensive, replicating to highly virulent, transmissive L. pneumophila is regulated. Copyright © 2017 Oliva et al.

Quantitative Assessment of the Heterogeneity of PD-L1 Expression in Non-Small-Cell Lung Cancer.

PubMed

McLaughlin, Joseph; Han, Gang; Schalper, Kurt A; Carvajal-Hausdorf, Daniel; Pelekanou, Vasiliki; Rehman, Jamaal; Velcheti, Vamsidhar; Herbst, Roy; LoRusso, Patricia; Rimm, David L

2016-01-01

Early-phase trials with monoclonal antibodies targeting PD-1 (programmed cell death protein 1) and PD-L1 (programmed cell death 1 ligand 1) have demonstrated durable clinical responses in patients with non-small-cell lung cancer (NSCLC). However, current assays for the prognostic and/or predictive role of tumor PD-L1 expression are not standardized with respect to either quantity or distribution of expression. To demonstrate PD-L1 protein distribution in NSCLC tumors using both conventional immunohistochemistry (IHC) and quantitative immunofluorescence (QIF) and compare results obtained using 2 different PD-L1 antibodies. PD-L1 was measured using E1L3N and SP142, 2 rabbit monoclonal antibodies, in 49 NSCLC whole-tissue sections and a corresponding tissue microarray with the same 49 cases. Non-small-cell lung cancer biopsy specimens from 2011 to 2012 were collected retrospectively from the Yale Thoracic Oncology Program Tissue Bank. Human melanoma Mel 624 cells stably transfected with PD-L1 as well as Mel 624 parental cells, and human term placenta whole tissue sections were used as controls and for antibody validation. PD-L1 protein expression in tumor and stroma was assessed using chromogenic IHC and the AQUA (Automated Quantitative Analysis) method of QIF. Tumor-infiltrating lymphocytes (TILs) were scored in hematoxylin-eosin slides using current consensus guidelines. The association between PD-L1 protein expression, TILs, and clinicopathological features were determined. PD-L1 expression discordance or heterogeneity using the diaminobenzidine chromogen and QIF was the main outcome measure selected prior to performing the study. Using chromogenic IHC, both antibodies showed fair to poor concordance. The PD-L1 antibodies showed poor concordance (Cohen κ range, 0.124-0.340) using conventional chromogenic IHC and showed intra-assay heterogeneity (E1L3N coefficient of variation [CV], 6.75%-75.24%; SP142 CV, 12.17%-109.61%) and significant interassay discordance using QIF (26.6%). Quantitative immunofluorescence showed that PD-L1 expression using both PD-L1 antibodies was heterogeneous. Using QIF, the scores obtained with E1L3N and SP142 for each tumor were significantly different according to nonparametric paired test (P < .001). Assessment of 588 serial section fields of view from whole tissue showed discordant expression at a frequency of 25%. Expression of PD-L1 was correlated with high TILs using both E1L3N (P = .007) and SP142 (P = .02). Objective determination of PD-L1 protein levels in NSCLC reveals heterogeneity within tumors and prominent interassay variability or discordance. This could be due to different antibody affinities, limited specificity, or distinct target epitopes. Efforts to determine the clinical value of these observations are under way.

Change in Minimum Orbit Intersection Distance due to General Relativistic Precession in Small Solar System Bodies

NASA Astrophysics Data System (ADS)

Sekhar, Aswin; Valsecchi, Giovanni B.; Asher, David; Werner, Stephanie; Vaubaillon, Jeremie; Li, Gongjie

2017-06-01

One of the greatest successes of Einstein's General Theory of Relativity (GR) was the correct prediction of the perihelion precession of Mercury. The closed form expression to compute this precession tells us that substantial GR precession would occur only if the bodies have a combination of both moderately small perihelion distance and semi-major axis. Minimum Orbit Intersection Distance (MOID) is a quantity which helps us to understand the closest proximity of two orbits in space. Hence evaluating MOID is crucial to understand close encounters and collision scenarios better. In this work, we look at the possible scenarios where a small GR precession in argument of pericentre can create substantial changes in MOID for small bodies ranging from meteoroids to comets and asteroids.Previous works have looked into neat analytical techniques to understand different collision scenarios and we use those standard expressions to compute MOID analytically. We find the nature of this mathematical function is such that a relatively small GR precession can lead to drastic changes in MOID values depending on the initial value of argument of pericentre. Numerical integrations were done with the MERCURY package incorporating GR code to test the same effects. A numerical approach showed the same interesting relationship (as shown by analytical theory) between values of argument of pericentre and the peaks or dips in MOID values. There is an overall agreement between both analytical and numerical methods.We find that GR precession could play an important role in the calculations pertaining to MOID and close encounter scenarios in the case of certain small solar system bodies (depending on their initial orbital elements) when long term impact risk possibilities are considered. Previous works have looked into impact probabilities and collision scenarios on planets from different small body populations. This work aims to find certain sub-sets of small bodies where GR could play an interesting role. Certain parallels are drawn between the cases of asteroids, comets and small perihelion distance meteoroid streams.

Intravenous infusion of hexamethonium and atropine but not propranolol diminishes apolipoprotein A-IV gene expression in rat ileum.

PubMed

Sonoyama, K; Tajima, K; Fujiwara, R; Kasai, T

2000-03-01

To clarify the role of neural factors in the regulation of apolipoprotein (apo) A-IV expression in the small intestine, we investigated the effect of neural blockers on mRNA levels of apo A-IV in rat small intestine. Either ganglionic blocker (hexamethonium), cholinergic blocker (atropine) or beta-adrenergic blocker (propranolol) was infused intravenously to unrestrained conscious rats for 8 h, and then total RNA was isolated from the small intestine and analyzed using Northern hybridization. Apo A-IV mRNA levels in the ileum were significantly lower in hexamethonium- or atropine-infused rats than in saline- (control) or propranolol-infused rats. Immunoblot analysis showed no difference in plasma apo A-IV concentrations between hexamethonium- and saline-infused groups. The lower mRNA levels of apo A-IV in the ileum of hexamethonium-infused rats were observed even in bile-drained rats, indicating that the lower expression was not due to any changes in bile availability. The ileal apo A-IV mRNA levels were significantly higher in rats infused with lipid emulsion into the ileum than in rats infused with glucose-saline, and the concomitant infusion of intravenous hexamethonium did not affect the higher levels of apo A-IV mRNA. These results suggest that the basal expression of the ileal A-IV gene is at least partially regulated in a site-specific manner by cholinergic neurons.

Molecular Phenotyping Small (Asian) versus Large (Western) Plaque Psoriasis Shows Common Activation of IL-17 Pathway Genes, but Different Regulatory Gene Sets

PubMed Central

Kim, Jaehwan; Oh, Chil-Hwan; Jeon, Jiehyun; Baek, Yoosang; Ahn, Jaewoo; Kim, Dong Joo; Lee, Hyun-Soo; da Rosa, Joel Correa; Suárez-Fariñas, Mayte; Lowes, Michelle A.; Krueger, James G.

2015-01-01

Psoriasis is present in all racial groups, but in varying frequencies and severity. Considering that small plaque psoriasis is specific to the Asian population and severe psoriasis is more predominant in the Western population, we defined Asian small and intermediate plaque psoriasis as psoriasis subtypes, and compared their molecular signatures with classic subtype of Western large plaque psoriasis. Two different characteristics of psoriatic spreading—vertical growth and radial expansion—were contrasted between subtypes, and genomic data were correlated to histologic and clinical measurements. Compared to Western large plaque psoriasis, Asian small plaque psoriasis revealed limited psoriasis spreading, but IL-17A and IL-17-regulated pro-inflammatory cytokines were highly expressed. Paradoxically, IL-17A and IL-17-regulated pro-inflammatory cytokines were lower in Western large plaque psoriasis, while T cells and dendritic cells in total psoriatic skin area were exponentially increased. Negative immune regulators, such as CD69 and FAS, were decreased in both Western large plaque psoriasis and psoriasis with accompanying arthritis or obesity, and their expression was correlated with psoriasis severity index. Based on the disease subtype comparisons, we propose that dysregulation of T cell expansion enabled by downregulation of immune negative regulators is the main mechanism for development of large plaque psoriasis subtypes. PMID:26763436

The Effect of DA-6034 on Intestinal Permeability in an Indomethacin-Induced Small Intestinal Injury Model.

PubMed

Kwak, Dong Shin; Lee, Oh Young; Lee, Kang Nyeong; Jun, Dae Won; Lee, Hang Lak; Yoon, Byung Chul; Choi, Ho Soon

2016-05-23

DA-6034 has anti-inflammatory activities and exhibits cytoprotective effects in acute gastric injury models. However, explanations for the protective effects of DA-6034 on intestinal permeability are limited. This study sought to investigate the effect of DA-6034 on intestinal permeability in an indomethacin-induced small intestinal injury model and its protective effect against small intestinal injury. Rats in the treatment group received DA-6034 from days 0 to 2 and indomethacin from days 1 to 2. Rats in the control group received indomethacin from days 1 to 2. On the fourth day, the small intestines were examined to compare the severity of inflammation. Intestinal permeability was evaluated by using fluorescein isothiocyanate-labeled dextran. Western blotting was performed to confirm the association between DA-6034 and the extracellular signal-regulated kinase (ERK) pathway. The inflammation scores in the treatment group were lower than those in the control group, but the difference was statistically insignificant. Hemorrhagic lesions in the treatment group were broader than those in the control group, but the difference was statistically insignificant. Intestinal permeability was lower in the treatment group than in the control group. DA-6034 enhanced extracellular signal-regulated kinase expression, and intestinal permeability was negatively correlated with ERK expression. DA-6034 may decrease intestinal permeability in an indomethacin-induced intestinal injury model via the ERK pathway.

Use of programmed cell death protein ligand 1 assay to predict the outcomes of non-small cell lung cancer patients treated with immune checkpoint inhibitors

PubMed Central

Tibaldi, Carmelo; Lunghi, Alice; Baldini, Editta

2017-01-01

The recent discovery of immune checkpoints inhibitors, especially anti-programmed cell death protein 1 (PD-1) and anti-programmed cell death protein ligand 1 (PD-L1) monoclonal antibodies, has opened new scenarios in the management of non-small cell lung cancer (NSCLC) and this new class of drugs has achieved a rapid development in the treatment of this disease. However, considering the costs of these drugs and the fact that only a subset of patients experience long-term disease control, the identification of predictive biomarkers for the selection of candidates suitable for treatment has become a priority. The research focused mainly on the expression of the PD-L1 receptor on both tumor cells and/or immune infiltrates determined by immunohistochemistry (IHC). However, different checkpoint inhibitors were tested, different IHC assays were used, different targets were considered (tumor cells, immune infiltrates or both) and different expression thresholds were employed in clinical trials. In some trials the assay was used prospectively to select the patients, while in other trials it was evaluated retrospectively. Some confusion emerges, which makes it difficult to easily compare the literature data and to translate them in practice management. This mini-review shows the possibilities and pitfalls of the PD-L1 expression to predict the activity and efficacy of anti PD1/PD-L1 monoclonal antibodies in the treatment of NSCLC. PMID:28848698

Disturbance of DKK1 level is partly involved in survival of lung cancer cells via regulation of ROMO1 and γ-radiation sensitivity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, In Gyu, E-mail: igkim@kaeri.re.kr; Department of Radiation Biotechnology and Applied Radioisotope, University of Science and Technology; Kim, Seo Yoen

2014-01-03

Highlights: •DKK1 was expressed differently among non-small-cell lung cancer cell lines. •DKK1 negatively regulated ROMO1 gene expression. •Disturbance of DKK1 level induced the imbalance of cellular ROS. •DKK1/ROMO1-induced ROS imbalance is involved in cell survival in NSCLC. -- Abstract: Dickkopf1 (DKK1), a secreted protein involved in embryonic development, is a potent inhibitor of the Wnt signaling pathway and has been postulated to be a tumor suppressor or tumor promoter depending on the tumor type. In this study, we showed that DKK1 was expressed differently among non-small-cell lung cancer cell lines. The DKK1 expression level was much higher in A549 cellsmore » than in H460 cells. We revealed that blockage of DKK1 expression by silencing RNA in A549 cells caused up-regulation of intracellular reactive oxygen species (ROS) modulator (ROMO1) protein, followed by partial cell death, cell growth inhibition, and loss of epithelial–mesenchymal transition property caused by ROS, and it also increased γ-radiation sensitivity. DKK1 overexpression in H460 significantly inhibited cell survival with the decrease of ROMO1 level, which induced the decrease of cellular ROS. Thereafter, exogenous N-acetylcysteine, an antioxidant, or hydrogen peroxide, a pro-oxidant, partially rescued cells from death and growth inhibition. In each cell line, both overexpression and blockage of DKK1 not only elevated p-RB activation, which led to cell growth arrest, but also inactivated AKT/NF-kB, which increased radiation sensitivity and inhibited cell growth. This study is the first to demonstrate that strict modulation of DKK1 expression in different cell types partially maintains cell survival via tight regulation of the ROS-producing ROMO1 and radiation resistance.« less

Comprehensive Expression Map of Transcription Regulators in the Adult Zebrafish Telencephalon Reveals Distinct Neurogenic Niches

PubMed Central

Diotel, Nicolas; Rodriguez Viales, Rebecca; Armant, Olivier; März, Martin; Ferg, Marco; Rastegar, Sepand; Strähle, Uwe

2015-01-01

The zebrafish has become a model to study adult vertebrate neurogenesis. In particular, the adult telencephalon has been an intensely studied structure in the zebrafish brain. Differential expression of transcriptional regulators (TRs) is a key feature of development and tissue homeostasis. Here we report an expression map of 1,202 TR genes in the telencephalon of adult zebrafish. Our results are summarized in a database with search and clustering functions to identify genes expressed in particular regions of the telencephalon. We classified 562 genes into 13 distinct patterns, including genes expressed in the proliferative zone. The remaining 640 genes displayed unique and complex patterns of expression and could thus not be grouped into distinct classes. The neurogenic ventricular regions express overlapping but distinct sets of TR genes, suggesting regional differences in the neurogenic niches in the telencephalon. In summary, the small telencephalon of the zebrafish shows a remarkable complexity in TR gene expression. The adult zebrafish telencephalon has become a model to study neurogenesis. We established the expression pattern of more than 1200 transcription regulators (TR) in the adult telencephalon. The neurogenic regions express overlapping but distinct sets of TR genes suggesting regional differences in the neurogenic potential. J. Comp. Neurol. 523:1202–1221, 2015. © 2015 Wiley Periodicals, Inc. PMID:25556858

Comprehensive expression map of transcription regulators in the adult zebrafish telencephalon reveals distinct neurogenic niches.

PubMed

Diotel, Nicolas; Rodriguez Viales, Rebecca; Armant, Olivier; März, Martin; Ferg, Marco; Rastegar, Sepand; Strähle, Uwe

2015-06-01

The zebrafish has become a model to study adult vertebrate neurogenesis. In particular, the adult telencephalon has been an intensely studied structure in the zebrafish brain. Differential expression of transcriptional regulators (TRs) is a key feature of development and tissue homeostasis. Here we report an expression map of 1,202 TR genes in the telencephalon of adult zebrafish. Our results are summarized in a database with search and clustering functions to identify genes expressed in particular regions of the telencephalon. We classified 562 genes into 13 distinct patterns, including genes expressed in the proliferative zone. The remaining 640 genes displayed unique and complex patterns of expression and could thus not be grouped into distinct classes. The neurogenic ventricular regions express overlapping but distinct sets of TR genes, suggesting regional differences in the neurogenic niches in the telencephalon. In summary, the small telencephalon of the zebrafish shows a remarkable complexity in TR gene expression. The adult zebrafish telencephalon has become a model to study neurogenesis. We established the expression pattern of more than 1200 transcription regulators (TR) in the adult telencephalon. The neurogenic regions express overlapping but distinct sets of TR genes suggesting regional differences in the neurogenic potential. © 2015 Wiley Periodicals, Inc.

Genome-Wide Identification and Comprehensive Expression Profiling of Ribosomal Protein Small Subunit (RPS) Genes and their Comparative Analysis with the Large Subunit (RPL) Genes in Rice

PubMed Central

Saha, Anusree; Das, Shubhajit; Moin, Mazahar; Dutta, Mouboni; Bakshi, Achala; Madhav, M. S.; Kirti, P. B.

2017-01-01

Ribosomal proteins (RPs) are indispensable in ribosome biogenesis and protein synthesis, and play a crucial role in diverse developmental processes. Our previous studies on Ribosomal Protein Large subunit (RPL) genes provided insights into their stress responsive roles in rice. In the present study, we have explored the developmental and stress regulated expression patterns of Ribosomal Protein Small (RPS) subunit genes for their differential expression in a spatiotemporal and stress dependent manner. We have also performed an in silico analysis of gene structure, cis-elements in upstream regulatory regions, protein properties and phylogeny. Expression studies of the 34 RPS genes in 13 different tissues of rice covering major growth and developmental stages revealed that their expression was substantially elevated, mostly in shoots and leaves indicating their possible involvement in the development of vegetative organs. The majority of the RPS genes have manifested significant expression under all abiotic stress treatments with ABA, PEG, NaCl, and H2O2. Infection with important rice pathogens, Xanthomonas oryzae pv. oryzae (Xoo) and Rhizoctonia solani also induced the up-regulation of several of the RPS genes. RPS4, 13a, 18a, and 4a have shown higher transcript levels under all the abiotic stresses, whereas, RPS4 is up-regulated in both the biotic stress treatments. The information obtained from the present investigation would be useful in appreciating the possible stress-regulatory attributes of the genes coding for rice ribosomal small subunit proteins apart from their functions as house-keeping proteins. A detailed functional analysis of independent genes is required to study their roles in stress tolerance and generating stress- tolerant crops. PMID:28966624

Expression of Kv3.1b potassium channel is widespread in macaque motor cortex pyramidal cells: A histological comparison between rat and macaque

PubMed Central

Soares, David; Goldrick, Isabelle; Lemon, Roger N.; Kraskov, Alexander; Greensmith, Linda

2017-01-01

Abstract There are substantial differences across species in the organization and function of the motor pathways. These differences extend to basic electrophysiological properties. Thus, in rat motor cortex, pyramidal cells have long duration action potentials, while in the macaque, some pyramidal neurons exhibit short duration “thin” spikes. These differences may be related to the expression of the fast potassium channel Kv3.1b, which in rat interneurons is associated with generation of thin spikes. Rat pyramidal cells typically lack these channels, while there are reports that they are present in macaque pyramids. Here we made a systematic, quantitative comparison of the Kv3.1b expression in sections from macaque and rat motor cortex, using two different antibodies (NeuroMab, Millipore). As our standard reference, we examined, in the same sections, Kv3.1b staining in parvalbumin‐positive interneurons, which show strong Kv3.1b immunoreactivity. In macaque motor cortex, a large sample of pyramidal neurons were nearly all found to express Kv3.1b in their soma membranes. These labeled neurons were identified as pyramidal based either by expression of SMI32 (a pyramidal marker), or by their shape and size, and lack of expression of parvalbumin (a marker for some classes of interneuron). Large (Betz cells), medium, and small pyramidal neurons all expressed Kv3.1b. In rat motor cortex, SMI32‐postive pyramidal neurons expressing Kv3.1b were very rare and weakly stained. Thus, there is a marked species difference in the immunoreactivity of Kv3.1b in pyramidal neurons, and this may be one of the factors explaining the pronounced electrophysiological differences between rat and macaque pyramidal neurons. PMID:28213922

Small RNA analysis in Petunia hybrida identifies unusual tissue-specific expression patterns of conserved miRNAs and of a 24mer RNA

PubMed Central

Tedder, Philip; Zubko, Elena; Westhead, David R.; Meyer, Peter

2009-01-01

Two pools of small RNAs were cloned from inflorescences of Petunia hybrida using a 5′-ligation dependent and a 5′-ligation independent approach. The two libraries were integrated into a public website that allows the screening of individual sequences against 359,769 unique clones. The library contains 15 clones with 100% identity and 53 clones with one mismatch to miRNAs described for other plant species. For two conserved miRNAs, miR159 and miR390, we find clear differences in tissue-specific distribution, compared with other species. This shows that evolutionary conservation of miRNA sequences does not necessarily include a conservation of the miRNA expression profile. Almost 60% of all clones in the database are 24-nucleotide clones. In accordance with the role of 24mers in marking repetitive regions, we find them distributed across retroviral and transposable element sequences but other 24mers map to promoter regions and to different transcript regions. For one target region we observe tissue-specific variation of matching 24mers, which demonstrates that, as for 21mers, 24mer concentrations are not necessarily identical in different tissues. Asymmetric distribution of a putative novel miRNA in the two libraries suggests that the cloning method can be selective for the representation of certain small RNAs in a collection. PMID:19369427

Small Heat Shock Protein Responses Differ between Chaparral Shrubs from Contrasting Microclimates

DOE PAGES

Knight, Charles A.

2010-01-01

Smore » mall heat shock protein (sHsp) responses were studied for two evergreen perennial shrubs in the northern California chaparral; one common on warm, south-facing slopes ( Ceanothus cuneatus ), and the other on cooler, north-facing slopes ( Prunus ilicifolia ). mall Hsp expression was induced experimentally for field collected leaves. Leaf collections were made where the species co-occur. mall Hsp expression was quantified using two antibodies, one specific to a chloroplast 22 kD sHsp and another that detects a broad range of sHsps. Differences between chloroplast sHsp accumulation, which protects thermally labile proteins in PII, and the general sHsp response were examined. The species from the cooler microclimate, Prunus , had a lower induction temperature and accumulated greater levels of sHsps at low temperatures. Both Prunus and Ceanothus reached peak sHsp expression at 42 ∘ C . The species from the warmer microclimate, Ceanothus , had greater sHsp expression at higher temperatures. Chloroplast sHsp expression generally tracked sHsp expression in Ceanothus , but in Prunus general Hsps were elevated before chloroplast sHsps. Variation between species for sHsp expression (induction temperatures, accumulation levels, and the duration of expression) coupled with the costs of Hsp synthesis, may contribute to differences in the abundance and distribution of plants across environmental gradients.« less

Glucose transporters and enzymes related to glucose synthesis in small intestinal mucosa of mid-lactation dairy cows fed 2 levels of starch.

PubMed

Lohrenz, A-K; Duske, K; Schönhusen, U; Losand, B; Seyfert, H M; Metges, C C; Hammon, H M

2011-09-01

Diets containing corn starch may improve glucose supply by providing significant amounts of intestinal starch and increasing intestinal glucose absorption in dairy cows. Glucose absorption in the small intestine requires specific glucose transporters; that is, sodium-dependent glucose co-transporter-1 (SGLT1) and facilitated glucose transporter (GLUT2), which are usually downregulated in the small intestine of functional ruminants but are upregulated when luminal glucose is available. We tested the hypothesis that mRNA and protein expression of intestinal glucose transporters and mRNA expression of enzymes related to gluconeogenesis are affected by variable starch supply. Dairy cows (n=9/group) were fed for 4 wk total mixed rations (TMR) containing either high (HS) or low (LS) starch levels in the diet. Feed intake and milk yield were measured daily. After slaughter, tissue samples of the small intestinal mucosa (mid-duodenum and mid-jejunum) were taken for determination of mRNA concentrations of SGLT1 and GLUT2 as well as pyruvate carboxylase, cytosolic phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase by real-time reverse transcription PCR relative to a housekeeping gene. Protein expression of GLUT2 in crude mucosal membranes and of SGLT1 and GLUT2 in brush-border membrane vesicles was quantified by sodium dodecyl sulfate-PAGE and immunoblot. A mixed model was used to examine feeding and time-related changes on feed intake and milk yield and to test feeding and gut site effects on gene or protein expression of glucose transporters and enzymes in the intestinal mucosa. Dry matter intake, but not energy intake, was higher in cows fed HS compared with LS. Abundance of SGLT1 mRNA tended to be higher in duodenal than in jejunal mucosa, and mRNA abundances of pyruvate carboxylase tended to be higher in jejunal than in duodenal mucosa. In brush-border membrane vesicles, SGLT1 and GLUT2 protein expression could be demonstrated. No diet-dependent differences were found concerning mRNA and protein contents of glucose transporter or mRNA level of gluconeogenic enzymes. In conclusion, our investigations on glucose transporters and gluconeogenic enzymes in the small intestinal mucosa of dairy cows did not show significant diet regulation when TMR with different amounts of intestinal starch were fed. Therefore, predicted intestinal glucose absorption after enhanced starch feeding is probably not supported by changes of intestinal glucose transporters in dairy cows. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Gravity-regulated gene expression in Arabidopsis thaliana

NASA Astrophysics Data System (ADS)

Sederoff, Heike; Brown, Christopher S.; Heber, Steffen; Kajla, Jyoti D.; Kumar, Sandeep; Lomax, Terri L.; Wheeler, Benjamin; Yalamanchili, Roopa

Plant growth and development is regulated by changes in environmental signals. Plants sense environmental changes and respond to them by modifying gene expression programs to ad-just cell growth, differentiation, and metabolism. Functional expression of genes comprises many different processes including transcription, translation, post-transcriptional and post-translational modifications, as well as the degradation of RNA and proteins. Recently, it was discovered that small RNAs (sRNA, 18-24 nucleotides long), which are heritable and systemic, are key elements in regulating gene expression in response to biotic and abiotic changes. Sev-eral different classes of sRNAs have been identified that are part of a non-cell autonomous and phloem-mobile network of regulators affecting transcript stability, translational kinetics, and DNA methylation patterns responsible for heritable transcriptional silencing (epigenetics). Our research has focused on gene expression changes in response to gravistimulation of Arabidopsis roots. Using high-throughput technologies including microarrays and 454 sequencing, we iden-tified rapid changes in transcript abundance of genes as well as differential expression of small RNA in Arabidopsis root apices after minutes of reorientation. Some of the differentially regu-lated transcripts are encoded by genes that are important for the bending response. Functional mutants of those genes respond faster to reorientation than the respective wild type plants, indicating that these proteins are repressors of differential cell elongation. We compared the gravity responsive sRNAs to the changes in transcript abundances of their putative targets and identified several potential miRNA: target pairs. Currently, we are using mutant and transgenic Arabidopsis plants to characterize the function of those miRNAs and their putative targets in gravitropic and phototropic responses in Arabidopsis.

Multiple Transduction Pathways Mediate Thyrotropin Receptor Signaling in Preosteoblast-Like Cells

PubMed Central

Boutin, Alisa; Neumann, Susanne

2016-01-01

It has been shown that the TSH receptor (TSHR) couples to a number of different signaling pathways, although the Gs-cAMP pathway has been considered primary. Here, we measured the effects of TSH on bone marker mRNA and protein expression in preosteoblast-like U2OS cells stably expressing TSHRs. We determined which signaling cascades are involved in the regulation of IL-11, osteopontin (OPN), and alkaline phosphatase (ALPL). We demonstrated that TSH-induced up-regulation of IL-11 is primarily mediated via the Gs pathway as IL-11 was up-regulated by forskolin (FSK), an adenylyl cyclase activator, and inhibited by protein kinase A inhibitor H-89 and by silencing of Gαs by small interfering RNA. OPN levels were not affected by FSK, but its up-regulation was inhibited by TSHR/Gi-uncoupling by pertussis toxin. Pertussis toxin decreased p38 MAPK kinase phosphorylation, and a p38 inhibitor and small interfering RNA knockdown of p38α inhibited OPN induction by TSH. Up-regulation of ALPL expression required high doses of TSH (EC50 = 395nM), whereas low doses (EC50 = 19nM) were inhibitory. FSK-stimulated cAMP production decreased basal ALPL expression, whereas protein kinase A inhibition by H-89 and silencing of Gαs increased basal levels of ALPL. Knockdown of Gαq/11 and a protein kinase C inhibitor decreased TSH-stimulated up-regulation of ALPL, whereas a protein kinase C activator increased ALPL levels. A MAPK inhibitor and silencing of ERK1/2 inhibited TSH-stimulated ALPL expression. We conclude that TSH regulates expression of different bone markers via distinct signaling pathways. PMID:26950201

Dicer-like 3 produces transposable element-associated 24-nt siRNAs that control agricultural traits in rice

PubMed Central

Wei, Liya; Gu, Lianfeng; Song, Xianwei; Cui, Xiekui; Lu, Zhike; Zhou, Ming; Wang, Lulu; Hu, Fengyi; Zhai, Jixian; Meyers, Blake C.; Cao, Xiaofeng

2014-01-01

Transposable elements (TEs) and repetitive sequences make up over 35% of the rice (Oryza sativa) genome. The host regulates the activity of different TEs by different epigenetic mechanisms, including DNA methylation, histone H3K9 methylation, and histone H3K4 demethylation. TEs can also affect the expression of host genes. For example, miniature inverted repeat TEs (MITEs), dispersed high copy-number DNA TEs, can influence the expression of nearby genes. In plants, 24-nt small interfering RNAs (siRNAs) are mainly derived from repeats and TEs. However, the extent to which TEs, particularly MITEs associated with 24-nt siRNAs, affect gene expression remains elusive. Here, we show that the rice Dicer-like 3 homolog OsDCL3a is primarily responsible for 24-nt siRNA processing. Impairing OsDCL3a expression by RNA interference caused phenotypes affecting important agricultural traits; these phenotypes include dwarfism, larger flag leaf angle, and fewer secondary branches. We used small RNA deep sequencing to identify 535,054 24-nt siRNA clusters. Of these clusters, ∼82% were OsDCL3a-dependent and showed significant enrichment of MITEs. Reduction of OsDCL3a function reduced the 24-nt siRNAs predominantly from MITEs and elevated expression of nearby genes. OsDCL3a directly targets genes involved in gibberellin and brassinosteroid homeostasis; OsDCL3a deficiency may affect these genes, thus causing the phenotypes of dwarfism and enlarged flag leaf angle. Our work identifies OsDCL3a-dependent 24-nt siRNAs derived from MITEs as broadly functioning regulators for fine-tuning gene expression, which may reflect a conserved epigenetic mechanism in higher plants with genomes rich in dispersed repeats or TEs. PMID:24554078

Rubisco small subunit, chlorophyll a/b-binding protein and sucrose:fructan-6-fructosyl transferase gene expression and sugar status in single barley leaf cells in situ. Cell type specificity and induction by light.

PubMed

Lu, Chungui; Koroleva, Olga A; Farrar, John F; Gallagher, Joe; Pollock, Chris J; Tomos, A Deri

2002-11-01

We describe a highly efficient two-step single-cell reverse transcriptase-polymerase chain reaction technique for analyzing gene expression at the single-cell level. Good reproducibility and a linear dose response indicated that the technique has high specificity and sensitivity for detection and quantification of rare RNA. Actin could be used as an internal standard. The expression of message for Rubisco small subunit (RbcS), chlorophyll a/b-binding protein (Cab), sucrose (Suc):fructan-6-fructosyl transferase (6-SFT), and Actin were measured in individual photosynthetic cells of the barley (Hordeum vulgare) leaf. Only Actin was found in the non-photosynthetic epidermal cells. Cab, RbcS, and 6-SFT genes were expressed at a low level in mesophyll and parenchymatous bundle sheath (BS) cells when sampled from plants held in dark for 40 h. Expression increased considerably after illumination. The amount of 6-SFT, Cab, and RbcS transcript increased more in mesophyll cells than in the parenchymatous BS cells. The difference may be caused by different chloroplast structure and posttranscriptional control in mesophyll and BS cells. When similar single-cell samples were assayed for Suc, glucose, and fructan, there was high correlation between 6-SFT gene expression and Suc and glucose concentrations. This is consistent with Suc concentration being the trigger for transcription. Together with earlier demonstrations that the mesophyll cells have a higher sugar threshold for fructan polymerization, our data may indicate separate control of transcription and enzyme activity. Values for the sugar concentrations of the individual cell types are reported.

Gender differences in emotion expression in children: a meta-analytic review.

PubMed

Chaplin, Tara M; Aldao, Amelia

2013-07-01

Emotion expression is an important feature of healthy child development that has been found to show gender differences. However, there has been no empirical review of the literature on gender and facial, vocal, and behavioral expressions of different types of emotions in children. The present study constitutes a comprehensive meta-analytic review of gender differences and moderators of differences in emotion expression from infancy through adolescence. We analyzed 555 effect sizes from 166 studies with a total of 21,709 participants. Significant but very small gender differences were found overall, with girls showing more positive emotions (g = -.08) and internalizing emotions (e.g., sadness, anxiety, sympathy; g = -.10) than boys, and boys showing more externalizing emotions (e.g., anger; g = .09) than girls. Notably, gender differences were moderated by age, interpersonal context, and task valence, underscoring the importance of contextual factors in gender differences. Gender differences in positive emotions were more pronounced with increasing age, with girls showing more positive emotions than boys in middle childhood (g = -.20) and adolescence (g = -.28). Boys showed more externalizing emotions than girls at toddler/preschool age (g = .17) and middle childhood (g = .13) and fewer externalizing emotions than girls in adolescence (g = -.27). Gender differences were less pronounced with parents and were more pronounced with unfamiliar adults (for positive emotions) and with peers/when alone (for externalizing emotions). Our findings of gender differences in emotion expression in specific contexts have important implications for gender differences in children's healthy and maladaptive development. 2013 APA, all rights reserved

Gender Differences in Emotion Expression in Children: A Meta-Analytic Review

PubMed Central

Chaplin, Tara M.; Aldao, Amelia

2012-01-01

Emotion expression is an important feature of healthy child development that has been found to show gender differences. However, there has been no empirical review of the literature on gender and facial, vocal, and behavioral expressions of different types of emotions in children. The present study constitutes a comprehensive meta-analytic review of gender differences, and moderators of differences, in emotion expression from infancy through adolescence. We analyzed 555 effect sizes from 166 studies with a total of 21,709 participants. Significant, but very small, gender differences were found overall, with girls showing more positive emotions (g = −.08) and internalizing emotions (e.g., sadness, anxiety, sympathy; g = −.10) than boys, and boys showing more externalizing emotions (e.g., anger; g = .09) than girls. Notably, gender differences were moderated by age, interpersonal context, and task valence, underscoring the importance of contextual factors in gender differences. Gender differences in positive emotions were more pronounced with increasing age, with girls showing more positive emotions than boys in middle childhood (g = −.20) and adolescence (g = −.28). Boys showed more externalizing emotions than girls at toddler/preschool age (g = .17) and middle childhood (g = .13) and fewer externalizing emotions than girls in adolescence (g = −.27). Gender differences were less pronounced with parents and were more pronounced with unfamiliar adults (for positive emotions) and with peers/when alone (for externalizing emotions). Our findings of gender differences in emotion expression in specific contexts have important implications for gender differences in children’s healthy and maladaptive development. PMID:23231534

The landscape of genomic imprinting across diverse adult human tissues

PubMed Central

Baran, Yael; Subramaniam, Meena; Biton, Anne; Tukiainen, Taru; Tsang, Emily K.; Rivas, Manuel A.; Pirinen, Matti; Gutierrez-Arcelus, Maria; Smith, Kevin S.; Kukurba, Kim R.; Zhang, Rui; Eng, Celeste; Torgerson, Dara G.; Urbanek, Cydney; Li, Jin Billy; Rodriguez-Santana, Jose R.; Burchard, Esteban G.; Seibold, Max A.; MacArthur, Daniel G.; Montgomery, Stephen B.; Zaitlen, Noah A.; Lappalainen, Tuuli

2015-01-01

Genomic imprinting is an important regulatory mechanism that silences one of the parental copies of a gene. To systematically characterize this phenomenon, we analyze tissue specificity of imprinting from allelic expression data in 1582 primary tissue samples from 178 individuals from the Genotype-Tissue Expression (GTEx) project. We characterize imprinting in 42 genes, including both novel and previously identified genes. Tissue specificity of imprinting is widespread, and gender-specific effects are revealed in a small number of genes in muscle with stronger imprinting in males. IGF2 shows maternal expression in the brain instead of the canonical paternal expression elsewhere. Imprinting appears to have only a subtle impact on tissue-specific expression levels, with genes lacking a systematic expression difference between tissues with imprinted and biallelic expression. In summary, our systematic characterization of imprinting in adult tissues highlights variation in imprinting between genes, individuals, and tissues. PMID:25953952

Expression and purification of moricin CM4 and human β-defensins 4 in Escherichia coli using a new technology.

PubMed

Shen, Yang; Ai, Hong-Xin; Song, Ren; Liang, Zhen-Ning; Li, Jian-Feng; Zhang, Shuang-Quan

2010-10-20

Different strategies have been developed to produce small antimicrobial peptides using recombinant techniques. Here we report a new technology of biosynthesis of moricin CM4 and human β-defensins 4 (HβD4) in the Escherichia coli. The CM4 and HβD4 gene were cloned into a vector containing the tags elastin-like peptide (ELP) and intein to construct the expression vector pET-EI-CM4 and pET-EI-HβD4. All the peptides, expressed as soluble fusions, were isolated from the protein debris by the method called inverse transition cycling (ITC) rather than traditional immobilized metal affinity chromatography (IMAC) and separated from the fusion leader by self-cleavage. Fully reduced peptides that were purified exhibited expected antimicrobial activity. The approach described here is a low-cost, convenient and potential way for generating small antimicrobial peptide. Copyright © 2010 Elsevier GmbH. All rights reserved.

Exposure to an extremely low-frequency electromagnetic field only slightly modifies the proteome of Chromobacterium violaceumATCC 12472.

PubMed

Baraúna, Rafael A; Santos, Agenor V; Graças, Diego A; Santos, Daniel M; Ghilardi, Rubens; Pimenta, Adriano M C; Carepo, Marta S P; Schneider, Maria P C; Silva, Artur

2015-05-01

Several studies of the physiological responses of different organisms exposed to extremely low-frequency electromagnetic fields (ELF-EMF) have been described. In this work, we report the minimal effects of in situ exposure to ELF-EMF on the global protein expression of Chromobacterium violaceum using a gel-based proteomic approach. The protein expression profile was only slightly altered, with five differentially expressed proteins detected in the exposed cultures; two of these proteins (DNA-binding stress protein, Dps, and alcohol dehydrogenase) were identified by MS/MS. The enhanced expression of Dps possibly helped to prevent physical damage to DNA. Although small, the changes in protein expression observed here were probably beneficial in helping the bacteria to adapt to the stress generated by the electromagnetic field.

Selective HDAC Inhibition for the Disruption of Latent HIV-1 Infection

PubMed Central

Barton, Kirston M.; Archin, Nancie M.; Keedy, Kara S.; Espeseth, Amy S.; Zhang, Yan-ling; Gale, Jennifer; Wagner, Florence F.; Holson, Edward B.; Margolis, David M.

2014-01-01

Selective histone deacetylase (HDAC) inhibitors have emerged as a potential anti-latency therapy for persistent human immunodeficiency virus type 1 (HIV-1) infection. We utilized a combination of small molecule inhibitors and short hairpin (sh)RNA-mediated gene knockdown strategies to delineate the key HDAC(s) to be targeted for selective induction of latent HIV-1 expression. Individual depletion of HDAC3 significantly induced expression from the HIV-1 promoter in the 2D10 latency cell line model. However, depletion of HDAC1 or −2 alone or in combination did not significantly induce HIV-1 expression. Co-depletion of HDAC2 and −3 resulted in a significant increase in expression from the HIV-1 promoter. Furthermore, concurrent knockdown of HDAC1, −2, and −3 resulted in a significant increase in expression from the HIV-1 promoter. Using small molecule HDAC inhibitors of differing selectivity to ablate the residual HDAC activity that remained after (sh)RNA depletion, the effect of depletion of HDAC3 was further enhanced. Enzymatic inhibition of HDAC3 with the selective small-molecule inhibitor BRD3308 activated HIV-1 transcription in the 2D10 cell line. Furthermore, ex vivo exposure to BRD3308 induced outgrowth of HIV-1 from resting CD4+ T cells isolated from antiretroviral-treated, aviremic HIV+ patients. Taken together these findings suggest that HDAC3 is an essential target to disrupt HIV-1 latency, and inhibition of HDAC2 may also contribute to the effort to purge and eradicate latent HIV-1 infection. PMID:25136952

Function of Protein Phosphatase 2A in Control of Proliferation: Isolation and Analysis of Dominant-Defective Mutants

DTIC Science & Technology

1999-06-01

subunits are expressed ubiquitously and appear to be encoded by small and quite homogeneous gene families. In plants , however, A and C subunit gene...1996). In both plants and animals, different B subunit isoforms are encoded by two or more unrelated gene families, some of which are expressed in a...PP2A functions in whole plants and in mammalian tissue culture cells. This genetic system may also prove useful for analyzing interactions between

RISC-Target Interaction: Cleavage and Translational Suppression

PubMed Central

van den Berg, Arjen; Mols, Johann; Han, Jiahuai

2008-01-01

Summary Small RNA molecules have been known and utilized to suppress gene expression for more than a decade. The discovery that these small RNA molecules are endogenously expressed in many organisms and have a critical role in controlling gene expression have led to the arising of a whole new field of research. Termed small interfering RNA (siRNA) or microRNA (miRNA) these ~22 nt RNA molecules have the capability to suppress gene expression through various mechanisms once they are incorporated in the multi-protein RNA-Induced Silencing Complex (RISC) and interact with their target mRNA. This review introduces siRNAs and microRNAs in a historical perspective and focuses on the key molecules in RISC, structural properties and mechanisms underlying the process of small RNA regulated post-transcriptional suppression of gene expression. PMID:18692607

Immune Cell Profiling of IFN-λ Response Shows pDCs Express Highest Level of IFN-λR1 and Are Directly Responsive via the JAK-STAT Pathway.

PubMed

Kelly, Aoife; Robinson, Mark W; Roche, Gerard; Biron, Christine A; O'Farrelly, Cliona; Ryan, Elizabeth J

2016-12-01

The interferon lambda (IFN-λ) cytokines have well-known antiviral properties, yet their contribution to immune regulation is not well understood. Epithelial cells represent the major target cell of IFN-λ; peripheral blood mononuclear cells are generally considered nonresponsive, with the exception of plasmacytoid dendritic cells (pDCs). In this study we aimed to define the potential for discrete subpopulations of cells to directly respond to IFN-λ. Analysis of peripheral blood leukocytes reveals that, while pDCs uniformly express the highest levels of IFN-λ receptor, a small proportion of B cells and monocytes also express the receptor. Nevertheless, B cells and monocytes respond poorly to IFN-λ stimulation in vitro, with minimal STAT phosphorylation and interferon-stimulated gene (ISG) induction observed. We confirm that pDCs respond to IFN-λ in vitro, upregulating their expression of pSTAT1, pSTAT3, and pSTAT5. However, we found that pDCs do not upregulate pSTAT6 in response to IFN-λ treatment. Our results highlight unique aspects of the response to IFN-λ and confirm that while the IFN-λ receptor is expressed by a small proportion of several different circulating immune cell lineages, under normal conditions only pDCs respond to IFN-λ stimulation with robust STAT phosphorylation and ISG induction. The difference in STAT6 responsiveness of pDCs to type I and type III interferons may help explain the divergence in their biological activities.

Stable isotope deltas: Tiny, yet robust signatures in nature

USGS Publications Warehouse

Brand, Willi A.; Coplen, Tyler B.

2012-01-01

Although most of them are relatively small, stable isotope deltas of naturally occurring substances are robust and enable workers in anthropology, atmospheric sciences, biology, chemistry, environmental sciences, food and drug authentication, forensic science, geochemistry, geology, oceanography, and paleoclimatology to study a variety of topics. Two fundamental processes explain the stable isotope deltas measured in most terrestrial systems: isotopic fractionation and isotope mixing. Isotopic fractionation is the result of equilibrium or kinetic physicochemical processes that fractionate isotopes because of small differences in physical or chemical properties of molecular species having different isotopes. It is shown that the mixing of radioactive and stable isotope end members can be modelled to provide information on many natural processes, including 14C abundances in the modern atmosphere and the stable hydrogen and oxygen isotopic compositions of the oceans during glacial and interglacial times. The calculation of mixing fractions using isotope balance equations with isotope deltas can be substantially in error when substances with high concentrations of heavy isotopes (e.g. 13C, 2H, and 18O ) are mixed. In such cases, calculations using mole fractions are preferred as they produce accurate mixing fractions. Isotope deltas are dimensionless quantities. In the International System of Units (SI), these quantities have the unit 1 and the usual list of prefixes is not applicable. To overcome traditional limitations with expressing orders of magnitude differences in isotope deltas, we propose the term urey (symbol Ur), after Harold C. Urey, for the unit 1. In such a manner, an isotope delta value expressed traditionally as−25 per mil can be written as−25 mUr (or−2.5 cUr or−0.25 dUr; the use of any SI prefix is possible). Likewise, very small isotopic differences often expressed in per meg ‘units’ are easily included (e.g. either+0.015 ‰ or+15 per meg can be written as+15 μUr.

Gene expression in lung adenocarcinomas of smokers and nonsmokers.

PubMed

Powell, Charles A; Spira, Avrum; Derti, Adnan; DeLisi, Charles; Liu, Gang; Borczuk, Alain; Busch, Steve; Sahasrabudhe, Sudhir; Chen, Yangde; Sugarbaker, David; Bueno, Raphael; Richards, William G; Brody, Jerome S

2003-08-01

Adenocarcinoma (AC) has become the most frequent type of lung cancer in men and women, and is the major form of lung cancer in nonsmokers. Our goal in this paper was to determine if AC in smokers and nonsmokers represents the same genetic disease. We compared gene expression profiles in resected samples of nonmalignant lung tissue and tumor tissue in six never-smokers with AC and in six smokers with AC, who were matched for clinical staging and histologic criteria of cell differentiation. Results were analyzed using a variety of bioinformatic tools. Four times as many genes changed expression in the transition from noninvolved lung to tumor in nonsmokers as in smokers, suggesting that AC in nonsmokers evolves locally, whereas AC in smokers evolves in a field of genetically altered tissue. There were some similarities in gene expression in smokers and nonsmokers, but many differences, suggesting different pathways of cell transformation and tumor formation. Gene expression in the noninvolved lungs of smokers differed from that of nonsmokers, and multidimensional scaling showed that noninvolved lungs of smokers groups with tumors rather than noninvolved lungs of nonsmokers. In addition, expression of a number of genes correlated with smoking intensity. Our findings, although limited by small sample size, suggest that additional studies comparing noninvolved to tumor tissue may identify pathogenetic mechanisms and therapeutic targets that differ in AC of smokers and nonsmokers.

Sequences 5' to translation start regulate expression of petunia rbcS genes.

PubMed Central

Dean, C; Favreau, M; Bedbrook, J; Dunsmuir, P

1989-01-01

The promoter sequences that contribute to quantitative differences in expression of the petunia genes (rbcS) encoding the small subunit of ribulose bisphosphate carboxylase have been characterized. The promoter regions of the two most abundantly expressed petunia rbcS genes, SSU301 and SSU611, show sequence similarity not present in other rbcS genes. We investigated the significance of these and other sequences by adding specific regions from the SSU301 promoter (the most strongly expressed gene) to equivalent regions in the SSU911 promoter (the least strongly expressed gene) and assaying the expression of the fusions in transgenic tobacco plants. In this way, we characterized an SSU301 promoter region (either from -285 to -178 or -291 to -204) which, when added to SSU911, in either orientation, increased SSU911 expression 25-fold. This increase was equivalent to that caused by addition of the entire SSU301 5'-flanking region. Replacement of SSU911 promoter sequences between -198 and the start codon with sequences from the equivalent region of SSU301 did not increase SSU911 expression significantly. The -291 to -204 SSU301 promoter fragment contributes significantly to quantitative differences in expression between the petunia rbcS genes. PMID:2535543

Identification of differentially expressed genes through RNA sequencing in goats (Capra hircus) at different postnatal stages

PubMed Central

Li, Qian; Lin, Sen

2017-01-01

Intramuscular fat (IMF) content and fatty acid composition of longissimus dorsi muscle (LM) change with growth, which partially determines the flavor and nutritional value of goat (Capra hircus) meat. However, unlike cattle, little information is available on the transcriptome-wide changes during different postnatal stages in small ruminants, especially goats. In this study, the sequencing reads of goat LM tissues collected from kid, youth, and adult period were mapped to the goat genome. Results showed that out of total 24 689 Unigenes, 20 435 Unigenes were annotated. Based on expected number of fragments per kilobase of transcript sequence per million base pairs sequenced (FPKM), 111 annotated differentially expressed genes (DEGs) were identified among different postnatal stages, which were subsequently assigned to 16 possible expression patterns by series-cluster analysis. Functional classification by Gene Ontology (GO) analysis was used for selecting the genes showing highest expression related to lipid metabolism. Finally, we identified the node genes for lipid metabolism regulation using co-expression analysis. In conclusion, these data may uncover candidate genes having functional roles in regulation of goat muscle development and lipid metabolism during the various growth stages in goats. PMID:28800357

Identification of differentially expressed genes through RNA sequencing in goats (Capra hircus) at different postnatal stages.

PubMed

Lin, Yaqiu; Zhu, Jiangjiang; Wang, Yong; Li, Qian; Lin, Sen

2017-01-01

Intramuscular fat (IMF) content and fatty acid composition of longissimus dorsi muscle (LM) change with growth, which partially determines the flavor and nutritional value of goat (Capra hircus) meat. However, unlike cattle, little information is available on the transcriptome-wide changes during different postnatal stages in small ruminants, especially goats. In this study, the sequencing reads of goat LM tissues collected from kid, youth, and adult period were mapped to the goat genome. Results showed that out of total 24 689 Unigenes, 20 435 Unigenes were annotated. Based on expected number of fragments per kilobase of transcript sequence per million base pairs sequenced (FPKM), 111 annotated differentially expressed genes (DEGs) were identified among different postnatal stages, which were subsequently assigned to 16 possible expression patterns by series-cluster analysis. Functional classification by Gene Ontology (GO) analysis was used for selecting the genes showing highest expression related to lipid metabolism. Finally, we identified the node genes for lipid metabolism regulation using co-expression analysis. In conclusion, these data may uncover candidate genes having functional roles in regulation of goat muscle development and lipid metabolism during the various growth stages in goats.

Predictive significance of bone sialoprotein and osteopontin for bone metastases in resected Chinese non-small-cell lung cancer patients: a large cohort retrospective study.

PubMed

Zhang, Li; Hou, Xue; Lu, Shun; Rao, Huilan; Hou, Jinghui; Luo, Rongzhen; Huang, He; Zhao, Hongyun; Jian, Hong; Chen, Zhiwei; Liao, Meilin; Wang, Xin

2010-01-01

Bone is one of the most common sites of metastasis in patients with non-small-cell lung cancer (NSCLC). Over-expression of bone sialoprotein (BSP) and osteopontin (OPN) in tumour samples has shown prognostic significance in bone metastasis (BM) of breast and prostate cancer, respectively. However, their importance in BM of NSCLC has not been verified. Therefore, we planned a large cohort retrospective study to investigate the relationship between the expression of these two biomarkers (BSP and OPN) and BM in surgically resected NSCLC patients. 180 completely resected NSCLC patients were included in this study. 40 patients subsequently developed BM. Paraffin-embedded primary tumour tissues of patients were supplied to produce a tissue microarray, and immunohistochemistry method was used for evaluation of the expression of BSP and OPN. Different expressions of these two biomarkers among BM group and non-BM group were estimated by chi(2) test. BM-free survival was analyzed by Kaplan-Meier method. The prognostic impact of clinicopathologic variables and biomarker expression was evaluated by Cox proportional hazards model. BSP expression was associated with BM (p=0.007), whereas OPN expression did not reach statistical significance (p=0.245). Univariate analysis showed that expression of BSP (p=0.010) and N staging (p<0.005) was associated with BM-free survival. Multivariate analyses showed BSP expression (HR=3.322, p=0.003), N staging (HR=1.879, p=0.001), and T staging (HR=1.618, p=0.024) were independent prognostic factors for BM. BSP protein expression in the primary resected NSCLC is strongly associated with BM and could be used to identify high-risk patients. Correlation of OPN protein expression and BM needs further investigation.

An integrated systems genetics screen reveals the transcriptional structure of inherited predisposition to metastatic disease

PubMed Central

Faraji, Farhoud; Hu, Ying; Wu, Gang; Goldberger, Natalie E.; Walker, Renard C.; Zhang, Jinghui; Hunter, Kent W.

2014-01-01

Metastasis is the result of stochastic genomic and epigenetic events leading to gene expression profiles that drive tumor dissemination. Here we exploit the principle that metastatic propensity is modified by the genetic background to generate prognostic gene expression signatures that illuminate regulators of metastasis. We also identify multiple microRNAs whose germline variation is causally linked to tumor progression and metastasis. We employ network analysis of global gene expression profiles in tumors derived from a panel of recombinant inbred mice to identify a network of co-expressed genes centered on Cnot2 that predicts metastasis-free survival. Modulating Cnot2 expression changes tumor cell metastatic potential in vivo, supporting a functional role for Cnot2 in metastasis. Small RNA sequencing of the same tumor set revealed a negative correlation between expression of the Mir216/217 cluster and tumor progression. Expression quantitative trait locus analysis (eQTL) identified cis-eQTLs at the Mir216/217 locus, indicating that differences in expression may be inherited. Ectopic expression of Mir216/217 in tumor cells suppressed metastasis in vivo. Finally, small RNA sequencing and mRNA expression profiling data were integrated to reveal that miR-3470a/b target a high proportion of network transcripts. In vivo analysis of Mir3470a/b demonstrated that both promote metastasis. Moreover, Mir3470b is a likely regulator of the Cnot2 network as its overexpression down-regulated expression of network hub genes and enhanced metastasis in vivo, phenocopying Cnot2 knockdown. The resulting data from this strategy identify Cnot2 as a novel regulator of metastasis and demonstrate the power of our systems-level approach in identifying modifiers of metastasis. PMID:24322557

Ion Channel Gene Expression in Lung Adenocarcinoma: Potential Role in Prognosis and Diagnosis

PubMed Central

Ko, Jae-Hong; Gu, Wanjun; Lim, Inja; Bang, Hyoweon; Ko, Eun A.; Zhou, Tong

2014-01-01

Ion channels are known to regulate cancer processes at all stages. The roles of ion channels in cancer pathology are extremely diverse. We systematically analyzed the expression patterns of ion channel genes in lung adenocarcinoma. First, we compared the expression of ion channel genes between normal and tumor tissues in patients with lung adenocarcinoma. Thirty-seven ion channel genes were identified as being differentially expressed between the two groups. Next, we investigated the prognostic power of ion channel genes in lung adenocarcinoma. We assigned a risk score to each lung adenocarcinoma patient based on the expression of the differentially expressed ion channel genes. We demonstrated that the risk score effectively predicted overall survival and recurrence-free survival in lung adenocarcinoma. We also found that the risk scores for ever-smokers were higher than those for never-smokers. Multivariate analysis indicated that the risk score was a significant prognostic factor for survival, which is independent of patient age, gender, stage, smoking history, Myc level, and EGFR/KRAS/ALK gene mutation status. Finally, we investigated the difference in ion channel gene expression between the two major subtypes of non-small cell lung cancer: adenocarcinoma and squamous-cell carcinoma. Thirty ion channel genes were identified as being differentially expressed between the two groups. We suggest that ion channel gene expression can be used to improve the subtype classification in non-small cell lung cancer at the molecular level. The findings in this study have been validated in several independent lung cancer cohorts. PMID:24466154

Spatial distributions of Kv4 channels and KChip2 isoforms in the murine heart based on laser capture microdissection.

PubMed

Teutsch, Christine; Kondo, Richard P; Dederko, Dorothy A; Chrast, Jacqueline; Chien, Kenneth R; Giles, Wayne R

2007-03-01

Regional differences in repolarizing K(+) current densities and expression levels of their molecular components are important for coordinating the pattern of electrical excitation and repolarization of the heart. The small size of hearts from mice may obscure these interventricular and/or transmural expression differences of K(+) channels. We have examined this possibility in adult mouse ventricle using a technology that provides very high spatial resolution of tissue collection. Conventional manual dissection and laser capture microdissection (LCM) were utilized to dissect tissue from distinct ventricular regions. RNA was isolated from epicardial, mid-myocardial and endocardial layers of both the right and left ventricles. Real-time RT-PCR was used to quantify the transcript expression in these different regions. LCM revealed significant interventricular and transmural gradients for both Kv4.2 and the alpha-subunit of KChIP2. The expression profile of a second K(+) channel transcript, Kir2.1, which is responsible for the inwardly rectifying K(+) current I(k1), showed no interventricular or transmural gradients and therefore served as a negative control. Our findings are in contrast to previous reports of a relatively uniform left ventricular transmural pattern of expression of Kv4.2, Kv4.3 and KChIP2 in adult mouse heart, which appear to be different than that in larger mammals. Specifically, our results demonstrate significant epi- to endocardial differences in the patterns of expression of both Kv4.2 and KChIP2.

The human neonatal small intestine has the potential for arginine synthesis; developmental changes in the expression of arginine-synthesizing and -catabolizing enzymes.

PubMed

Köhler, Eleonore S; Sankaranarayanan, Selvakumari; van Ginneken, Christa J; van Dijk, Paul; Vermeulen, Jacqueline L M; Ruijter, Jan M; Lamers, Wouter H; Bruder, Elisabeth

2008-11-10

Milk contains too little arginine for normal growth, but its precursors proline and glutamine are abundant; the small intestine of rodents and piglets produces arginine from proline during the suckling period; and parenterally fed premature human neonates frequently suffer from hypoargininemia. These findings raise the question whether the neonatal human small intestine also expresses the enzymes that enable the synthesis of arginine from proline and/or glutamine. Carbamoylphosphate synthetase (CPS), ornithine aminotransferase (OAT), argininosuccinate synthetase (ASS), arginase-1 (ARG1), arginase-2 (ARG2), and nitric-oxide synthase (NOS) were visualized by semiquantitative immunohistochemistry in 89 small-intestinal specimens. Between 23 weeks of gestation and 3 years after birth, CPS- and ASS-protein content in enterocytes was high and then declined to reach adult levels at 5 years. OAT levels declined more gradually, whereas ARG-1 was not expressed. ARG-2 expression increased neonatally to adult levels. Neurons in the enteric plexus strongly expressed ASS, OAT, NOS1 and ARG2, while varicose nerve fibers in the circular layer of the muscularis propria stained for ASS and NOS1 only. The endothelium of small arterioles expressed ASS and NOS3, while their smooth-muscle layer expressed OAT and ARG2. The human small intestine acquires the potential to produce arginine well before fetuses become viable outside the uterus. The perinatal human intestine therefore resembles that of rodents and pigs. Enteral ASS behaves as a typical suckling enzyme because its expression all but disappears in the putative weaning period of human infants.

The human neonatal small intestine has the potential for arginine synthesis; developmental changes in the expression of arginine-synthesizing and -catabolizing enzymes

PubMed Central

Köhler, Eleonore S; Sankaranarayanan, Selvakumari; van Ginneken, Christa J; van Dijk, Paul; Vermeulen, Jacqueline LM; Ruijter, Jan M; Lamers, Wouter H; Bruder, Elisabeth

2008-01-01

Background Milk contains too little arginine for normal growth, but its precursors proline and glutamine are abundant; the small intestine of rodents and piglets produces arginine from proline during the suckling period; and parenterally fed premature human neonates frequently suffer from hypoargininemia. These findings raise the question whether the neonatal human small intestine also expresses the enzymes that enable the synthesis of arginine from proline and/or glutamine. Carbamoylphosphate synthetase (CPS), ornithine aminotransferase (OAT), argininosuccinate synthetase (ASS), arginase-1 (ARG1), arginase-2 (ARG2), and nitric-oxide synthase (NOS) were visualized by semiquantitative immunohistochemistry in 89 small-intestinal specimens. Results Between 23 weeks of gestation and 3 years after birth, CPS- and ASS-protein content in enterocytes was high and then declined to reach adult levels at 5 years. OAT levels declined more gradually, whereas ARG-1 was not expressed. ARG-2 expression increased neonatally to adult levels. Neurons in the enteric plexus strongly expressed ASS, OAT, NOS1 and ARG2, while varicose nerve fibers in the circular layer of the muscularis propria stained for ASS and NOS1 only. The endothelium of small arterioles expressed ASS and NOS3, while their smooth-muscle layer expressed OAT and ARG2. Conclusion The human small intestine acquires the potential to produce arginine well before fetuses become viable outside the uterus. The perinatal human intestine therefore resembles that of rodents and pigs. Enteral ASS behaves as a typical suckling enzyme because its expression all but disappears in the putative weaning period of human infants. PMID:19000307

Endogenous small RNAs and antibacterial immunity in plants.

PubMed

Jin, Hailing

2008-08-06

Small RNAs are non-coding regulatory RNA molecules that control gene expression by mediating mRNA degradation, translational inhibition, or chromatin modification. Virus-derived small RNAs induce silencing of viral RNAs and are essential for antiviral defense in both animal and plant systems. The role of host endogenous small RNAs on antibacterial immunity has only recently been recognized. Host disease resistance and defense responses are achieved by activation and repression of a large array of genes. Certain endogenous small RNAs in plants, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are induced or repressed in response to pathogen attack and subsequently regulate the expression of genes involved in disease resistance and defense responses by mediating transcriptional or post-transcriptional gene silencing. Thus, these small RNAs play an important role in gene expression reprogramming in plant disease resistance and defense responses. This review focuses on the recent findings of plant endogenous small RNAs in antibacterial immunity.

New basic approach to treat non-small cell lung cancer based on RNA-interference.

PubMed

Makowiecki, Christina; Nolte, Andrea; Sutaj, Besmire; Keller, Timea; Avci-Adali, Meltem; Stoll, Heidi; Schlensak, Christian; Wendel, Hans Peter; Walker, Tobias

2014-03-01

To date the therapy for non-small cell lung cancer (NSCLC) is associated with severe side effects, frustrating outcomes, and does not consider different tumor characteristics. The RNA-interference (RNAi) pathway represents a potential new approach to treat NSCLC. With small interfering ribonucleic acids (siRNAs), it is possible to reduce the expression of proliferation-dependent proteins in tumor cells, leading to their apoptosis. We propose that siRNAs could be adapted to the tumor type and may cause fewer side effects than current therapy. Four NSCLC cell lines were cultured under standard conditions and transfected with three different concentrations of siRNAs targeted against the hypoxia-inducible factors 1α and 2α (HIF1α and HIF2α) and signal transducer and activator of transcription 3 (STAT3). The expression was observed by quantitative real-time polymerase chain reaction and western blots. For the analysis of cell growth three days after transfection, the cell number was detected using a CASY cell counter system. The results of the silencing of the analyzed factors differ in each cell line. Cell growth was significantly reduced in all cell lines after transfection with HIF1α- and STAT3-siRNA. The silencing of HIF2α resulted in a significant effect on cell growth in squamous, and large-cell lung cancer. This study shows that the knockdown and viability to siRNA transfection differ in each tumor type according to the used siRNA. This implies that the tumor types differ among themselves and should be treated differently. Therefore, the authors suggest a possible approach to a more personalized treatment of NSCLC.

Chaperones are necessary for the expression of catalytically active potato apyrases in prokaryotic cells.

PubMed

Porowińska, Dorota; Czarnecka, Joanna; Komoszyński, Michał

2014-07-01

NTPDases (nucleoside triphosphate diphosphohydrolases) (also called in plants apyrases) hydrolyze nucleoside 5'-tri- and/or diphosphate bonds producing nucleosides di or monophosphate and inorganic phosphate. For years, studies have been carried out to use both plant and animal enzymes for medicine. Therefore, there is a need to develop an efficient method for the quick production of large amounts of homogeneous proteins with high catalytic activity. Expression of proteins in prokaryotic cells is the most common way for the protein production. The aim of our study was to develop a method of expression of potato apyrase (StAPY4, 5, and 6) genes in bacterial cells under conditions that allowed the production of catalytically active form of these enzymes. Apyrase 4 and 6 were overexpressed in BL21-CodonPlus (DE3) bacteria strain but they were accumulated in inclusion bodies, regardless of the culture conditions and induction method. Co-expression of potato apyrases with molecular chaperones allowed the expression of catalytically active apyrase 5. However, its high nucleotidase activity could be toxic for bacteria and is therefore synthesized in small amounts in cells. Our studies show that each protein requires other conditions for maturation and even small differences in amino acid sequence can essentially affect protein folding regardless of presence of chaperones.

Identifying Stress Transcription Factors Using Gene Expression and TF-Gene Association Data

PubMed Central

Wu, Wei-Sheng; Chen, Bor-Sen

2007-01-01

Unicellular organisms such as yeasts have evolved to survive environmental stresses by rapidly reorganizing the genomic expression program to meet the challenges of harsh environments. The complex adaptation mechanisms to stress remain to be elucidated. In this study, we developed Stress Transcription Factor Identification Algorithm (STFIA), which integrates gene expression and TF-gene association data to identify the stress transcription factors (TFs) of six kinds of stresses. We identified some general stress TFs that are in response to various stresses, and some specific stress TFs that are in response to one specific stress. The biological significance of our findings is validated by the literature. We found that a small number of TFs may be sufficient to control a wide variety of expression patterns in yeast under different stresses. Two implications can be inferred from this observation. First, the adaptation mechanisms to different stresses may have a bow-tie structure. Second, there may exist extensive regulatory cross-talk among different stress responses. In conclusion, this study proposes a network of the regulators of stress responses and their mechanism of action. PMID:20066130

Gene Expression Patterns in Peripheral Blood Leukocytes in Patients with Recurrent Ciguatera Fish Poisoning: Preliminary Studies.

PubMed

Lopez, Maria-Cecilia; Ungaro, Ricardo F; Baker, Henry V; Moldawer, Lyle L; Robertson, Alison; Abbott, Margaret; Roberts, Sparkle M; Grattan, Lynn M; Morris, J Glenn

2016-07-01

Ciguatera fish poisoning (ciguatera) is a common clinical syndrome in areas where there is dependence on tropical reef fish for food. A subset of patients develops recurrent and, in some instances, chronic symptoms, which may result in substantial disability. To identify possible biomarkers for recurrent/chronic disease, and to explore correlations with immune gene expression, peripheral blood leukocyte gene expression in 10 ciguatera patients (7 recurrent, 3 acute) from the U.S. Virgin Islands, and 5 unexposed Florida controls were evaluated. Significant differences in gene expression were noted when comparing ciguatera patients and controls; however, it was not possible to differentiate between patients with acute and recurrent disease, possibly due to the small sample sizes involved.

Gene Expression Patterns in Peripheral Blood Leukocytes in Patients with Recurrent Ciguatera Fish Poisoning: Preliminary Studies

PubMed Central

Lopez, Maria-Cecilia; Ungaro, Ricardo F.; Baker, Henry V.; Moldawer, Lyle L.; Robertson, Alison; Abbott, Margaret; Roberts, Sparkle M.; Grattan, Lynn M.; Morris, J. Glenn

2016-01-01

Ciguatera fish poisoning (ciguatera) is a common clinical syndrome in areas where there is dependence on tropical reef fish for food. A subset of patients develops recurrent and, in some instances, chronic symptoms, which may result in substantial disability. To identify possible biomarkers for recurrent/chronic disease, and to explore correlations with immune gene expression, peripheral blood leukocyte gene expression in 10 ciguatera patients (7 recurrent, 3 acute) from the U.S. Virgin Islands, and 5 unexposed Florida controls were evaluated. Significant differences in gene expression were noted when comparing ciguatera patients and controls; however, it was not possible to differentiate between patients with acute and recurrent disease, possibly due to the small sample sizes involved. PMID:27594814

The Molecular Detection and Clinical Significance of ALK Rearrangement in Selected Advanced Non-Small Cell Lung Cancer: ALK Expression Provides Insights into ALK Targeted Therapy

PubMed Central

Zhang, Ning-Ning; Liu, Yu-Tao; Ma, Li; Wang, Lin; Hao, Xue-Zhi; Yuan, Zheng; Lin, Dong-Mei; Li, Dan; Zhou, Yu-Jie; Lin, Hua; Han, Xiao-Hong; Sun, Yan; Shi, Yuankai

2014-01-01

Background This study aimed to elucidate clinical significance of anaplastic lymphoma kinase (ALK) rearrangement in selected advanced non-small cell lung cancer (NSCLC), to compare the application of different ALK detection methods, and especially evaluate a possible association between ALK expression and clinical outcomes in crizotinib-treated patients. Methods ALK status was assessed by fluorescent in situ hybridization (FISH), immunohistochemistry (IHC) and quantitative RT-PCR (qRT-PCR) in 173 selected advanced NSCLC patients. Clinicopathologic data, genotype status and survival outcomes were analyzed. Moreover, the association of ALK expression with clinical outcomes was evaluated in ALK FISH-positive crizotinib-treated patients including two patients with concurrent epidermal growth factor receptor (EGFR) mutation. Results The positivity detection rate of ALK rearrangement by FISH, IHC and qRT-PCR was 35.5% (59/166), 35.7% (61/171), and 27.9% (34/122), respectively. ALK rearrangement was observed predominantly in young patients, never or light smokers, and adenocarcinomas, especially with signet ring cell features and poor differentiation. Median progression-free survival (PFS) of crizotinib-treated patients was 7.6 months. The overall survival (OS) of these patients was longer compared with that of crizotinib-naive or wild-type cohorts, but there was no significant difference in OS compared with patients with EGFR mutation. ALK expression did not associate with PFS; but, when ALK expression was analyzed as a dichotomous variable, moderate and strong ALK expression had a decreased risk of death (P = 0.026). The two patients with concomitant EGFR and ALK alterations showed difference in ALK expression, response to EGFR and ALK inhibitors, and overall survival. Conclusions Selective enrichment according to clinicopathologic features in NSCLC patients could highly improve the positivity detection rate of ALK rearrangement for ALK-targeted therapy. IHC could provide more clues for clinical trial design and therapeutic strategies for ALK-positive NSCLC patients including patients with double genetic aberration of ALK and EGFR. PMID:24404167

Growth of embryo and gene expression of nutrient transporters in the small intestine of the domestic pigeon (Columba livia).

PubMed

Chen, Ming-xia; Li, Xiang-guang; Yang, Jun-xian; Gao, Chun-qi; Wang, Bin; Wang, Xiu-qi; Yan, Hui-chao

2015-06-01

The objective of this study was to investigate the relationship between gene expression of nutrient (amino acid, peptide, sodium and proton) transporters in the small intestine and embryonic growth in domestic pigeons (Columba livia). One hundred and twenty-five fertilized eggs were randomly assigned into five groups and were incubated under optimal conditions (temperature of 38.1 °C and relative humidity of 55%). Twenty embryos/birds from each group were sacrificed by cervical dislocation on embryonic day (E) 9, 11, 13, 15 and day of hatch (DOH). The eggs, embryos (without yolk sac), and organs (head, brain, heart, liver, lungs, kidney, gizzard, small intestine, legs, and thorax) were dissected, cleaned, and weighed. Small intestine samples were collected for RNA isolation. The mRNA abundance of intestinal nutrient transporters was evaluated by real-time reverse transcription-polymerase chain reaction (RT-PCR). We classified these ten organs into four types according to the changes in relative weight during embryonic development. In addition, the gene expression of nutrient transporters was differentially regulated by embryonic day. The mRNA abundances of b(0,+)AT, EAAT3, y(+)LAT2, PepT1, LAT4, NHE2, and NHE3 increased linearly with age, whereas mRNA abundances of CAT1, CAT2, LAT1, EAAT2, SNAT1, and SNAT2 were increased to higher levels on E9 or E11 and then decreased to lower levels until DOH. The results of correlation analysis showed that the gene expressions of b(0,+)AT, EAAT3, PepT1, LAT4, NHE2, NHE3, and y(+)LAT2 had positive correlations with body weight (0.71

Analysis of H3K27me3 expression and DNA methylation at CCGG sites in smoking and non-smoking patients with non-small cell lung cancer and their clinical significance

PubMed Central

Zhu, Kunshou; Deng, Yujie; Weng, Guoxing; Hu, Dan; Huang, Cheng; Matsumoto, Keitaro; Nagayasu, Takeshi; Koji, Takehiko; Zheng, Xiongwei; Jiang, Wenhui; Lin, Gen; Cai, Yibin; Weng, Guibin; Chen, Xiaohui

2018-01-01

Smoking frequently leads to epigenetic alterations, including DNA methylation and histone modifications. The effect that smoking has on the DNA methylation levels at CCGG sites, the expression of trimethylation of histone H3 at lysine 27 (H3K27me3) and enhancer of zeste homolog 2 (EZH2), and their interactions in patients with non-small cell lung cancer (NSCLC) were analyzed. There were a total of 42 patients with NSCLC, 22 with adenocarcinomas and 20 with squamous cell carcinomas enrolled in the present study. Expression of H3K27me3, EZH2 and proliferating cellular nuclear antigen (PCNA) were immunohistochemically detected. DNA methylation at CCGG sites was evaluated via histoendonuclease-linked detection of DNA methylation sites. The apoptotic index of cancerous tissues obtained from patients of different smoking statuses was evaluated via the terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick end labeling method. The association with clinicopathological data was calculated relative to different smoking statuses. Compared with the non-smokers, smokers with NSCLC exhibited a significantly lower apoptotic index (P<0.05), and frequently had a lower level of DNA methylation at CCGG sites, lower H3K27me3 expression and a higher EZH2 expression (P<0.05). DNA methylation levels at CCGG sites were negatively correlated to the Brinkman index (P=0.017). Furthermore, there was a parallel association between the H3K27me3 and EZH2 expression levels in the majority of smokers, whereas in the majority of non-smokers, there was a diverging association (P=0.015). There was a diverging association between the PCNA and EZH2 expression levels in the majority of smokers; however, in the majority of non-smokers, there was a parallel association (P=0.048). In addition, the association between the CCGG methylation ratio and immunohistochemical expression of H3K27me3 was a parallel association in the majority of smokers, while in the majority of non-smokers there was a diverging association (P=0.049). Conclusively, patients with NSCLC and different smoking statuses exhibit different epigenetic characteristics. Additionally, DNA methylation levels at the CCGG sites may have the ability to determine associations between the expression levels of H3K27me3, EZH2 and PCNA. PMID:29616099

Use of mRNA expression signatures to discover small molecule inhibitors of skeletal muscle atrophy

PubMed Central

Adams, Christopher M.; Ebert, Scott M.; Dyle, Michael C.

2017-01-01

Purpose of review Here, we discuss a recently developed experimental strategy for discovering small molecules with potential to prevent and treat skeletal muscle atrophy. Recent findings Muscle atrophy involves and requires widespread changes in skeletal muscle gene expression, which generate complex but measurable patterns of positive and negative changes in skeletal muscle mRNA levels (a.k.a. mRNA expression signatures of muscle atrophy). Many bioactive small molecules generate their own characteristic mRNA expression signatures, and by identifying small molecules whose signatures approximate mirror images of muscle atrophy signatures, one may identify small molecules with potential to prevent and/or reverse muscle atrophy. Unlike a conventional drug discovery approach, this strategy does not rely on a predefined molecular target but rather exploits the complexity of muscle atrophy to identify small molecules that counter the entire spectrum of pathological changes in atrophic muscle. We discuss how this strategy has been used to identify two natural compounds, ursolic acid and tomatidine, that reduce muscle atrophy and improve skeletal muscle function. Summary Discovery strategies based on mRNA expression signatures can elucidate new approaches for preserving and restoring muscle mass and function. PMID:25807353

Use of mRNA expression signatures to discover small molecule inhibitors of skeletal muscle atrophy.

PubMed

Adams, Christopher M; Ebert, Scott M; Dyle, Michael C

2015-05-01

Here, we discuss a recently developed experimental strategy for discovering small molecules with potential to prevent and treat skeletal muscle atrophy. Muscle atrophy involves and requires widespread changes in skeletal muscle gene expression, which generate complex but measurable patterns of positive and negative changes in skeletal muscle mRNA levels (a.k.a. mRNA expression signatures of muscle atrophy). Many bioactive small molecules generate their own characteristic mRNA expression signatures, and by identifying small molecules whose signatures approximate mirror images of muscle atrophy signatures, one may identify small molecules with potential to prevent and/or reverse muscle atrophy. Unlike a conventional drug discovery approach, this strategy does not rely on a predefined molecular target but rather exploits the complexity of muscle atrophy to identify small molecules that counter the entire spectrum of pathological changes in atrophic muscle. We discuss how this strategy has been used to identify two natural compounds, ursolic acid and tomatidine, that reduce muscle atrophy and improve skeletal muscle function. Discovery strategies based on mRNA expression signatures can elucidate new approaches for preserving and restoring muscle mass and function.

Circadian rhythms and light responsiveness of mammalian clock gene, Clock and BMAL1, transcripts in the rat retina.

PubMed

Namihira, M; Honma, S; Abe, H; Tanahashi, Y; Ikeda, M; Honma, K

1999-08-13

Circadian expression and light-responsiveness of the mammalian clock genes, Clock and BMAL1, in the rat retina were examined by in situ hydbribization under constant darkness. A small but significant daily variation was detected in the Clock transcript level, but not in BMAL1. Light increased the Clock and BMAL1 expressions significantly when examined 60 min after exposure. The light-induced gene expression was phase-dependent for Clock and peaked at ZT2, while rather constant throughout the day for BMAL1. These findings suggest that Clock and BMAL1 play different roles in the generation of circadian rhytm in the retina from those in the suprachiasmatic nucleus. Different roles are also suggested between the two genes in the photic signal transduction in the retina.

Expression of small leucine-rich extracellular matrix proteoglycans biglycan and lumican reveals oral lichen planus malignant potential.

PubMed

Lončar-Brzak, Božana; Klobučar, Marko; Veliki-Dalić, Irena; Sabol, Ivan; Kraljević Pavelić, Sandra; Krušlin, Božo; Mravak-Stipetić, Marinka

2018-03-01

The aim of this study was to examine molecular alterations on the protein level in lesions of oral lichen planus (OLP), oral squamous cell carcinoma (OSCC) and healthy mucosa. Global protein profiling methods based on liquid chromatography coupled to mass spectrometry (LC-MS) were used, with a special emphasis on evaluation of deregulated extracellular matrix molecules expression, as well as on analyses of IG2F and IGFR2 expression in healthy mucosa, OLP and OSCC tissues by comparative semi-quantitative immunohistochemistry. Mass spectrometry-based proteomics profiling of healthy mucosa, OLP and OSCC tissues (and accompanied histologically unaltered tissues, respectively) identified 55 extracellular matrix proteins. Twenty among identified proteins were common to all groups of samples. Expression of small leucine-rich extracellular matrix proteoglycans lumican and biglycan was found both in OSCC and OLP and they were validated by Western blot analysis as putative biomarkers. A significant increase (p < 0.05) of biglycan expression in OLP-AT group was determined in comparison with OLP-T group, while lumican showed significant up-regulation (p < 0.05) in OLP-T and OSCC-T groups vs. adjacent and control tissue groups. Biglycan expression was only determined in OSCC-AT group. Immunohistochemical analysis of IGF2 and IG2FR expression revealed no significant difference among groups of samples. Biglycan and lumican were identified as important pathogenesis biomarkers of OLP that point to its malignant potential.

Effect of Dietary Nutrient Density on Small Intestinal Phosphate Transport and Bone Mineralization of Broilers during the Growing Period.

PubMed

Li, Jianhui; Yuan, Jianmin; Miao, Zhiqiang; Song, Zhigang; Yang, Yu; Tian, Wenxia; Guo, Yuming

2016-01-01

A 2 × 4 factorial experiment was conducted to determine the effects of dietary nutrient density on growth performance, small intestinal epithelial phosphate transporter expression, and bone mineralization of broiler chicks fed with diets with different nutrient densities and nonphytate phosphorus (NPP) levels. The broilers were fed with the same starter diets from 0 to 21 days of age. In the grower phase (day 22 to 42), the broilers were randomly divided into eight groups according to body weight. Relatively high dietary nutrient density (HDND) and low dietary nutrient density (LDND) diets were assigned metabolic energy (ME) values of 3,150 and 2,950 kcal/kg, respectively. Crude protein and essential amino acid levels were maintained in the same proportion as ME to prepare the two diet types. NPP levels were 0.25%, 0.30%, 0.35%, and 0.40% of the diets. Results showed that a HDND diet significantly increased the body weight gain (BWG) of broilers and significantly decreased the feed conversion ratio and NPP consumed per BWG. HDND significantly decreased tibial P content of the broilers. Conversely, mRNA expression of NaPi-IIb and protein expression of calbindin were significantly increased in the intestine of broilers fed a HDND diet. HDND also increased vitamin D receptor (VDR) expression, especially at a relatively low dietary NPP level (0.25%). The mRNA expression of NaPi-IIa in the kidneys was significantly increased at a relatively low dietary NPP level (0.25%) to maintain P balance. Tibial P, calcium, and ash content were significantly decreased, as were calbindin and VDR expression levels in the intestine at a low NPP level. Therefore, HDND improved the growth rate of broilers and increased the expression of phosphate and calcium transporter in the small intestine, but adversely affected bone mineralization.

Time Course of Substance P Expression in Dorsal Root Ganglia Following Complete Spinal Nerve Transection

PubMed Central

Weissner, Wendy; Winterson, Barbara J.; Stuart-Tilley, Alan; Devor, Marshall; Bove, Geoffrey M.

2008-01-01

Recent evidence suggests that substance P (SP) is upregulated in primary sensory neurons following axotomy, and that this change occurs in larger neurons that do not usually produce SP. If so, this upregulation may allow normally neighboring, uninjured, and non-nociceptive dorsal root ganglion (DRG) neurons to become effective in activating pain pathways. Using immunohistochemistry, we performed a unilateral L5 spinal nerve transection upon male Wistar rats, and measured SP expression in ipsilateral L4 and L5 DRGs and contralateral L5 DRGs, at 1 to 14 days postoperatively (dpo), and in control and sham operated rats. In normal and sham operated DRGs, SP was detectable almost exclusively in small neurons (≤ 800 μm2). Following surgery, the mean size of SP-positive neurons from the axotomized L5 ganglia was greater at 2, 4, 7 and 14 dpo. Among large neurons (> 800 μm2) from the axotomized L5, the percentage of SP-positive neurons increased at 2, 4, 7, and 14 dpo. Among small neurons from the axotomized L5, the percentage of SP-positive neurons was increased at 1 and 3 dpo, but was decreased at 7 and 14 dpo. Thus, SP expression is affected by axonal damage, and the time course of the expression is different between large and small DRG neurons. These data support a role of SP-producing, large DRG neurons in persistent sensory changes due to nerve injury. PMID:16680762

Mesenchymal Stem Cells Suppress Chronic Rejection in Heterotopic Small Intestine Transplant Rat Models Via Inhibition of CD68, Transforming Growth Factor- β1, and Platelet-Derived Growth Factor Expression.

PubMed

Li, Fuxin; Cao, Jisen; Zhao, Zhicheng; Li, Chuan; Qi, Feng; Liu, Tong

2017-04-01

Mesenchymal stem cells are easy to obtain and expand, with characteristics of low immunogenicity and strong tissue repair capacity. In this study, our aim was to investigate the role of mesenchymal stem cells in chronic immune rejection of heterotopic small intestine transplant in rats. After successfully constructing a rat chronic immune rejection model of heterotopic small intestine transplant, we infused mesenchymal stem cells into the animal recipients. We observed mesenchymal stem cell location in the recipients, recipient survival, pathology changes, and the expression of CD68, transforming growth factor β1, and platelet-derived growth factor C in the donor intestine. Mesenchymal stem cells inhibited the lymphocyte proliferation caused by concanavalin A in vitro. After stem cells were infused into recipients, they were mainly located in the donor intestine, as well as in the spleen and thymus. Recovery after transplant and pathology changes of the donor intestine in rats with stem cell infusion were better than in the control group; however, we observed no differences in survival time, accompanied by downregulated expression of CD68, transforming growth factor β1, and platelet-derived growth factor C. Mesenchymal stem cells, to a certain extent, could inhibit the process of chronic rejection. The mechanisms may include the inhibited function of these cells on lymphocyte proliferation, reduced infiltration of macrophages, and reduced expression of transforming growth factor β1 and platelet-derived growth factor C.

[PD-L1 expression: An emerging biomarker in non-small cell lung cancer].

PubMed

Adam, Julien; Planchard, David; Marabelle, Aurélien; Soria, Jean-Charles; Scoazec, Jean-Yves; Lantuéjoul, Sylvie

2016-01-01

Therapies targeting immune checkpoints, in particular programmed death 1 (PD-1) and its ligand programmed death ligand 1 (PD-L1), are major new strategies for the treatment of several malignancies including mestatatic non-small cell lung cancer (NSCLC). The identification of predictive biomarkers of response is required, considering efficacy, cost and potential adverse events. Expression of PD-L1 by immunohistochemistry has been associated with higher response rate and overall survival in several clinical trials evaluating anti-PD-1 and anti-PD-L1 monoclonal antibodies. Thus, PD-L1 immunohistochemical companion assays could be required for treatment with some of these therapies in NSCLC. However, heterogeneity in methodologies of PD-L1 assays in terms of primary antibodies and scoring algorithms, and tumor heterogenity for PD-L1 expression are important issues to be considered. More studies are required to compare the different assays, ensure their harmonization and standardization and identify the optimal conditions for testing. PD-L1 expression is likely an imperfect predictive biomarker for patient selection and association with other markers of the tumor immune microenvironment will be probably necessary in the future. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

GENE-Counter: A Computational Pipeline for the Analysis of RNA-Seq Data for Gene Expression Differences

PubMed Central

Di, Yanming; Schafer, Daniel W.; Wilhelm, Larry J.; Fox, Samuel E.; Sullivan, Christopher M.; Curzon, Aron D.; Carrington, James C.; Mockler, Todd C.; Chang, Jeff H.

2011-01-01

GENE-counter is a complete Perl-based computational pipeline for analyzing RNA-Sequencing (RNA-Seq) data for differential gene expression. In addition to its use in studying transcriptomes of eukaryotic model organisms, GENE-counter is applicable for prokaryotes and non-model organisms without an available genome reference sequence. For alignments, GENE-counter is configured for CASHX, Bowtie, and BWA, but an end user can use any Sequence Alignment/Map (SAM)-compliant program of preference. To analyze data for differential gene expression, GENE-counter can be run with any one of three statistics packages that are based on variations of the negative binomial distribution. The default method is a new and simple statistical test we developed based on an over-parameterized version of the negative binomial distribution. GENE-counter also includes three different methods for assessing differentially expressed features for enriched gene ontology (GO) terms. Results are transparent and data are systematically stored in a MySQL relational database to facilitate additional analyses as well as quality assessment. We used next generation sequencing to generate a small-scale RNA-Seq dataset derived from the heavily studied defense response of Arabidopsis thaliana and used GENE-counter to process the data. Collectively, the support from analysis of microarrays as well as the observed and substantial overlap in results from each of the three statistics packages demonstrates that GENE-counter is well suited for handling the unique characteristics of small sample sizes and high variability in gene counts. PMID:21998647

Small RNAs of Sequoia sempervirens during rejuvenation and phase change.

PubMed

Chen, Y-T; Shen, C-H; Lin, W-D; Chu, H-A; Huang, B-L; Kuo, C-I; Yeh, K-W; Huang, L-C; Chang, I-F

2013-01-01

In this work, the population of small RNAs (sRNAs) was studied in the gymnosperm Sequoia sempervirens during phase changes, specifically in the juvenile, adult and rejuvenated plants obtained in vitro. The potential target genes of Sequoia sRNAs were predicted through bioinformatics. Rejuvenation is a pivotal process in woody plants that enables them to regain their growth potential, which results in the recovery of physiologic and molecular characteristics that were lost when the juveniles mature into adult plants. The results from the five repeated graftings of juvenile, adult and rejuvenated plants in vitro showed that sRNAs could be classified into structural RNAs (Group I), small interfering RNAs (Group II), annotated microRNAs (Group III, and unannotated sRNAs (Group IV). The results indicate that only 573 among 15,485,415 sRNAs (Groups III and IV) had significantly different expression patterns associated with rejuvenation and phase change. A total of 215 sRNAs exhibited up-regulated expression patterns in adult shoots, and 358 sRNAs were down-regulated. Expression profiling and prediction of possible target genes of these unique small RNAs indicate possible functions in the control of photosynthetic efficiency and rooting competence abundance during plant rejuvenation. Moreover, the increase in SsmiR156 and decrease in SsmiR172 during plant rejuvenation suggested that these two microRNAs extensively affect phase transition. © 2012 German Botanical Society and The Royal Botanical Society of the Netherlands.

Small, synthetic, GC-rich mRNA stem-loop modules 5' proximal to the AUG start-codon predictably tune gene expression in yeast.

PubMed

Lamping, Erwin; Niimi, Masakazu; Cannon, Richard D

2013-07-29

A large range of genetic tools has been developed for the optimal design and regulation of complex metabolic pathways in bacteria. However, fewer tools exist in yeast that can precisely tune the expression of individual enzymes in novel metabolic pathways suitable for industrial-scale production of non-natural compounds. Tuning expression levels is critical for reducing the metabolic burden of over-expressed proteins, the accumulation of toxic intermediates, and for redirecting metabolic flux from native pathways involving essential enzymes without negatively affecting the viability of the host. We have developed a yeast membrane protein hyper-expression system with critical advantages over conventional, plasmid-based, expression systems. However, expression levels are sometimes so high that they adversely affect protein targeting/folding or the growth and/or phenotype of the host. Here we describe the use of small synthetic mRNA control modules that allowed us to predictably tune protein expression levels to any desired level. Down-regulation of expression was achieved by engineering small GC-rich mRNA stem-loops into the 5' UTR that inhibited translation initiation of the yeast ribosomal 43S preinitiation complex (PIC). Exploiting the fact that the yeast 43S PIC has great difficulty scanning through GC-rich mRNA stem-loops, we created yeast strains containing 17 different RNA stem-loop modules in the 5' UTR that expressed varying amounts of the fungal multidrug efflux pump reporter Cdr1p from Candida albicans. Increasing the length of mRNA stem-loops (that contained only GC-pairs) near the AUG start-codon led to a surprisingly large decrease in Cdr1p expression; ~2.7-fold for every additional GC-pair added to the stem, while the mRNA levels remained largely unaffected. An mRNA stem-loop of seven GC-pairs (∆G = -15.8 kcal/mol) reduced Cdr1p expression levels by >99%, and even the smallest possible stem-loop of only three GC-pairs (∆G = -4.4 kcal/mol) inhibited Cdr1p expression by ~50%. We have developed a simple cloning strategy to fine-tune protein expression levels in yeast that has many potential applications in metabolic engineering and the optimization of protein expression in yeast. This study also highlights the importance of considering the use of multiple cloning-sites carefully to preclude unwanted effects on gene expression.

Small, synthetic, GC-rich mRNA stem-loop modules 5′ proximal to the AUG start-codon predictably tune gene expression in yeast

PubMed Central

2013-01-01

Background A large range of genetic tools has been developed for the optimal design and regulation of complex metabolic pathways in bacteria. However, fewer tools exist in yeast that can precisely tune the expression of individual enzymes in novel metabolic pathways suitable for industrial-scale production of non-natural compounds. Tuning expression levels is critical for reducing the metabolic burden of over-expressed proteins, the accumulation of toxic intermediates, and for redirecting metabolic flux from native pathways involving essential enzymes without negatively affecting the viability of the host. We have developed a yeast membrane protein hyper-expression system with critical advantages over conventional, plasmid-based, expression systems. However, expression levels are sometimes so high that they adversely affect protein targeting/folding or the growth and/or phenotype of the host. Here we describe the use of small synthetic mRNA control modules that allowed us to predictably tune protein expression levels to any desired level. Down-regulation of expression was achieved by engineering small GC-rich mRNA stem-loops into the 5′ UTR that inhibited translation initiation of the yeast ribosomal 43S preinitiation complex (PIC). Results Exploiting the fact that the yeast 43S PIC has great difficulty scanning through GC-rich mRNA stem-loops, we created yeast strains containing 17 different RNA stem-loop modules in the 5′ UTR that expressed varying amounts of the fungal multidrug efflux pump reporter Cdr1p from Candida albicans. Increasing the length of mRNA stem-loops (that contained only GC-pairs) near the AUG start-codon led to a surprisingly large decrease in Cdr1p expression; ~2.7-fold for every additional GC-pair added to the stem, while the mRNA levels remained largely unaffected. An mRNA stem-loop of seven GC-pairs (∆G = −15.8 kcal/mol) reduced Cdr1p expression levels by >99%, and even the smallest possible stem-loop of only three GC-pairs (∆G = −4.4 kcal/mol) inhibited Cdr1p expression by ~50%. Conclusion We have developed a simple cloning strategy to fine-tune protein expression levels in yeast that has many potential applications in metabolic engineering and the optimization of protein expression in yeast. This study also highlights the importance of considering the use of multiple cloning-sites carefully to preclude unwanted effects on gene expression. PMID:23895661

Behavioral analysis of Drosophila transformants expressing human taste receptor genes in the gustatory receptor neurons.

PubMed

Adachi, Ryota; Sasaki, Yuko; Morita, Hiromi; Komai, Michio; Shirakawa, Hitoshi; Goto, Tomoko; Furuyama, Akira; Isono, Kunio

2012-06-01

Transgenic Drosophila expressing human T2R4 and T2R38 bitter-taste receptors or PKD2L1 sour-taste receptor in the fly gustatory receptor neurons and other tissues were prepared using conventional Gal4/UAS binary system. Molecular analysis showed that the transgene mRNAs are expressed according to the tissue specificity of the Gal4 drivers. Transformants expressing the transgene taste receptors in the fly taste neurons were then studied by a behavioral assay to analyze whether transgene chemoreceptors are functional and coupled to the cell response. Since wild-type flies show strong aversion against the T2R ligands as in mammals, the authors analyzed the transformants where the transgenes are expressed in the fly sugar receptor neurons so that they promote feeding ligand-dependently if they are functional and activate the neurons. Although the feeding preference varied considerably among different strains and individuals, statistical analysis using large numbers of transformants indicated that transformants expressing T2R4 showed a small but significant increase in the preference for denatonium and quinine, the T2R4 ligands, as compared to the control flies, whereas transformants expressing T2R38 did not. Similarly, transformants expressing T2R38 and PKD2L1 also showed a similar preference increase for T2R38-specific ligand phenylthiocarbamide (PTC) and a sour-taste ligand, citric acid, respectively. Taken together, the transformants expressing mammalian taste receptors showed a small but significant increase in the feeding preference that is taste receptor and also ligand dependent. Although future improvements are required to attain performance comparable to the endogenous robust response, Drosophila taste neurons may serve as a potential in vivo heterologous expression system for analyzing chemoreceptor function.

Decreased activity and expression of intestinal oligopeptide transporter PEPT1 in rats with hyperthyroidism in vivo.

PubMed

Ashida, Kayoko; Katsura, Toshiya; Saito, Hideyuki; Inui, Ken-ichi

2004-06-01

To examine the effect of thyroid hormone status on PEPT1 in vivo, the activity and expression of PEPT1 in the small intestine were examined in euthyroid and hyperthyroid rats. Hyperthyroidism was induced by treating rats with L-thyroxine (12 mg/L) in the drinking water for 21 days. Transport activity was measured by everted small intestinal preparations and in situ intestinal loop technique. Expressions of PEPT1 mRNA and protein were evaluated by competitive polymerase chain reaction and Western blotting, respectively. The uptake of [14C]glycylsarcosine by everted small intestinal preparations was significantly decreased in hyperthyroid rats, whereas that of methyl-alpha-D-[14C(U)]-glucopyranoside was not altered. Kinetic analysis showed that the Vmax value for [14C]glycylsarcosine uptake was significantly decreased in hyperthyroid rats, whereas the Km value was not affected. The mean portal vein concentrations after intrajejunal administration of [14C]glycylsarcosine were also decreased in hyperthyroid rats. Moreover, hyperthyroidism caused a significant decrease in the expression of PEPT1 mRNA in the small intestine, whereas the expression of Na+/glucose cotransporter (SGLT1) mRNA was not changed. The level of PEPT1 protein was also decreased in the small intestine of hyperthyroid rats. These results indicate that in hyperthyroid rats, the activity and expression of PEPT1 were decreased in the small intestine.

Arsenite and its metabolites, MMA(III) and DMA(III), modify CYP3A4, PXR and RXR alpha expression in the small intestine of CYP3A4 transgenic mice.

PubMed

Medina-Díaz, I M; Estrada-Muñiz, E; Reyes-Hernández, O D; Ramírez, P; Vega, L; Elizondo, G

2009-09-01

Arsenic is an environmental pollutant that has been associated with an increased risk for the development of cancer and several other diseases through alterations of cellular homeostasis and hepatic function. Cytochrome P450 (P450) modification may be one of the factors contributing to these disorders. Several reports have established that exposure to arsenite modifies P450 expression by decreasing or increasing mRNA and protein levels. Cytochrome P450 3A4 (CYP3A4), the predominant P450 expressed in the human liver and intestines, which is regulated mainly by the Pregnane X Receptor-Retinoid X Receptor alpha (PXR-RXR alpha) heterodimer, contributes to the metabolism of approximately half the drugs in clinical use today. The present study investigates the effect of sodium arsenite and its metabolites monomethylarsonous acid (MMA(III)) and dimethylarsinous acid (DMA(III)) on CYP3A4, PXR, and RXR alpha expression in the small intestine of CYP3A4 transgenic mice. Sodium arsenite treatment increases mRNA, protein and CYP3A4 activity in a dose-dependent manner. However, the increase in protein expression was not as marked as compared to the increase in mRNA levels. Arsenite treatment induces the accumulation of Ub-protein conjugates, indicating that the activation of this mechanism may explain the differences observed between the mRNA and protein expression of CYP3A4 induction. Treatment with 0.05 mg/kg of DMA(III) induces CYP3A4 in a similar way, while treatment with 0.05 mg/kg of MMA(III) increases mostly mRNA, and to a lesser degree, CYP3A4 activity. Sodium arsenite and both its metabolites increase PXR mRNA, while only DMA(III) induces RXR alpha expression. Overall, these results suggest that sodium arsenite and its metabolites induce CYP3A4 expression by increasing PXR expression in the small intestine of CYP3A4 transgenic mice.

Arsenite and its metabolites, MMA{sup III} and DMA{sup III}, modify CYP3A4, PXR and RXR alpha expression in the small intestine of CYP3A4 transgenic mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Medina-Diaz, I.M.; Estrada-Muniz, E.; Reyes-Hernandez, O.D.

Arsenic is an environmental pollutant that has been associated with an increased risk for the development of cancer and several other diseases through alterations of cellular homeostasis and hepatic function. Cytochrome P450 (P450) modification may be one of the factors contributing to these disorders. Several reports have established that exposure to arsenite modifies P450 expression by decreasing or increasing mRNA and protein levels. Cytochrome P450 3A4 (CYP3A4), the predominant P450 expressed in the human liver and intestines, which is regulated mainly by the Pregnane X Receptor-Retinoid X Receptor alpha (PXR-RXR alpha) heterodimer, contributes to the metabolism of approximately half themore » drugs in clinical use today. The present study investigates the effect of sodium arsenite and its metabolites monomethylarsonous acid (MMA{sup III}) and dimethylarsinous acid (DMA{sup III}) on CYP3A4, PXR, and RXR alpha expression in the small intestine of CYP3A4 transgenic mice. Sodium arsenite treatment increases mRNA, protein and CYP3A4 activity in a dose-dependent manner. However, the increase in protein expression was not as marked as compared to the increase in mRNA levels. Arsenite treatment induces the accumulation of Ub-protein conjugates, indicating that the activation of this mechanism may explain the differences observed between the mRNA and protein expression of CYP3A4 induction. Treatment with 0.05 mg/kg of DMA{sup III} induces CYP3A4 in a similar way, while treatment with 0.05 mg/kg of MMA{sup III} increases mostly mRNA, and to a lesser degree, CYP3A4 activity. Sodium arsenite and both its metabolites increase PXR mRNA, while only DMA{sup III} induces RXR alpha expression. Overall, these results suggest that sodium arsenite and its metabolites induce CYP3A4 expression by increasing PXR expression in the small intestine of CYP3A4 transgenic mice.« less

A novel highly differentially expressed gene in wheat endosperm associated with bread quality

PubMed Central

Furtado, A.; Bundock, P. C.; Banks, P. M.; Fox, G.; Yin, X.; Henry, R. J.

2015-01-01

Analysis of gene expression in developing wheat seeds was used to identify a gene, wheat bread making (wbm), with highly differential expression (~1000 fold) in the starchy endosperm of genotypes varying in bread making quality. Several alleles differing in the 5’-upstream region (promoter) of this gene were identified, with one present only in genotypes with high levels of wbm expression. RNA-Seq analysis revealed low or no wbm expression in most genotypes but high expression (0.2-0.4% of total gene expression) in genotypes that had good bread loaf volume. The wbm gene is predicted to encode a mature protein of 48 amino acids (including four cysteine residues) not previously identified in association with wheat quality, possibly because of its small size and low frequency in the wheat gene pool. Genotypes with high wbm expression all had good bread making quality but not always good physical dough qualities. The predicted protein was sulphur rich suggesting the possibility of a contribution to bread loaf volume by supporting the crossing linking of proteins in gluten. Improved understanding of the molecular basis of differences in bread making quality may allow more rapid development of high performing genotypes with acceptable end-use properties and facilitate increased wheat production. PMID:26011437

A novel highly differentially expressed gene in wheat endosperm associated with bread quality.

PubMed

Furtado, A; Bundock, P C; Banks, P M; Fox, G; Yin, X; Henry, R J

2015-05-26

Analysis of gene expression in developing wheat seeds was used to identify a gene, wheat bread making (wbm), with highly differential expression (~1000 fold) in the starchy endosperm of genotypes varying in bread making quality. Several alleles differing in the 5'-upstream region (promoter) of this gene were identified, with one present only in genotypes with high levels of wbm expression. RNA-Seq analysis revealed low or no wbm expression in most genotypes but high expression (0.2-0.4% of total gene expression) in genotypes that had good bread loaf volume. The wbm gene is predicted to encode a mature protein of 48 amino acids (including four cysteine residues) not previously identified in association with wheat quality, possibly because of its small size and low frequency in the wheat gene pool. Genotypes with high wbm expression all had good bread making quality but not always good physical dough qualities. The predicted protein was sulphur rich suggesting the possibility of a contribution to bread loaf volume by supporting the crossing linking of proteins in gluten. Improved understanding of the molecular basis of differences in bread making quality may allow more rapid development of high performing genotypes with acceptable end-use properties and facilitate increased wheat production.

Import routes and nuclear functions of Argonaute and other small RNA-silencing proteins.

PubMed

Schraivogel, Daniel; Meister, Gunter

2014-09-01

Small RNAs are important regulators of gene expression in many different organisms. Nuclear and cytoplasmic biogenesis enzymes generate functional small RNAs from double-stranded (ds) or single-stranded (ss) RNA precursors, and mature small RNAs are loaded into Argonaute proteins. In the cytoplasm, small RNAs guide Argonaute proteins to complementary RNAs leading to cleavage of these targets, translational silencing, or mRNA decay. In the nucleus Argonaute proteins engage in transcriptional silencing processes such as epigenetic silencing of repetitive elements at the chromatin level. During the past few years many novel functions of small RNA-guided gene silencing proteins in the nucleus have been reported. However, their specific import routes are largely unknown. In this review we summarize the current knowledge on nuclear transport routes that Argonaute and other RNA-silencing proteins take to carry out their various functions in the nucleus. Copyright © 2014 Elsevier Ltd. All rights reserved.

Exposure to an extremely low-frequency electromagnetic field only slightly modifies the proteome of Chromobacterium violaceumATCC 12472

PubMed Central

Baraúna, Rafael A.; Santos, Agenor V.; Graças, Diego A.; Santos, Daniel M.; Ghilardi, Rubens; Pimenta, Adriano M. C.; Carepo, Marta S. P.; Schneider, Maria P.C.; Silva, Artur

2015-01-01

Several studies of the physiological responses of different organisms exposed to extremely low-frequency electromagnetic fields (ELF-EMF) have been described. In this work, we report the minimal effects of in situ exposure to ELF-EMF on the global protein expression of Chromobacterium violaceum using a gel-based proteomic approach. The protein expression profile was only slightly altered, with five differentially expressed proteins detected in the exposed cultures; two of these proteins (DNA-binding stress protein, Dps, and alcohol dehydrogenase) were identified by MS/MS. The enhanced expression of Dps possibly helped to prevent physical damage to DNA. Although small, the changes in protein expression observed here were probably beneficial in helping the bacteria to adapt to the stress generated by the electromagnetic field. PMID:26273227

Identification and characterisation of side population cells in the canine pituitary gland.

PubMed

van Rijn, Sarah J; Gremeaux, Lies; Riemers, Frank M; Brinkhof, Bas; Vankelecom, Hugo; Penning, Louis C; Meij, Björn P

2012-06-01

To date, stem/progenitor cells have not been identified in the canine pituitary gland. Cells that efficiently exclude the vital dye Hoechst 33342 can be visualised and identified using fluorescence activated cell sorting (FACS) as a 'side population' (SP), distinct from the main population (MP). Such SPs have been identified in several tissues and display stem/progenitor cell characteristics. In this study, a small SP (1.3%, n=6) was detected in the anterior pituitary glands of healthy dogs. Quantitative PCR indicated significantly higher expression of CD34 and Thy1 in this SP, but no differences in the expression of CD133, Bmi-1, Axin2 or Shh. Pro-opiomelanocortin (POMC) and Lhx3 expression were significantly higher in the MP than in the SP, but no differences in the expression of Tpit, GH or PRL were found. The study demonstrated the existence of an SP of cells in the normal canine pituitary gland, encompassing cells with stem cell characteristics and without POMC expression. Copyright © 2011 Elsevier Ltd. All rights reserved.

The low noise limit in gene expression

DOE PAGES

Dar, Roy D.; Weinberger, Leor S.; Cox, Chris D.; ...

2015-10-21

Protein noise measurements are increasingly used to elucidate biophysical parameters. Unfortunately noise analyses are often at odds with directly measured parameters. Here we show that these inconsistencies arise from two problematic analytical choices: (i) the assumption that protein translation rate is invariant for different proteins of different abundances, which has inadvertently led to (ii) the assumption that a large constitutive extrinsic noise sets the low noise limit in gene expression. While growing evidence suggests that transcriptional bursting may set the low noise limit, variability in translational bursting has been largely ignored. We show that genome-wide systematic variation in translational efficiencymore » can-and in the case of E. coli does-control the low noise limit in gene expression. Therefore constitutive extrinsic noise is small and only plays a role in the absence of a systematic variation in translational efficiency. Lastly, these results show the existence of two distinct expression noise patterns: (1) a global noise floor uniformly imposed on all genes by expression bursting; and (2) high noise distributed to only a select group of genes.« less

Identification of neoblast- and regeneration-specific miRNAs in the planarian Schmidtea mediterranea

PubMed Central

Sasidharan, Vidyanand; Lu, Yi-Chien; Bansal, Dhiru; Dasari, Pranavi; Poduval, Deepak; Seshasayee, Aswin; Resch, Alissa M.; Graveley, Brenton R.; Palakodeti, Dasaradhi

2013-01-01

In recent years, the planarian Schmidtea mediterranea has emerged as a tractable model system to study stem cell biology and regeneration. MicroRNAs are small RNA species that control gene expression by modulating translational repression and mRNA stability and have been implicated in the regulation of various cellular processes. Though recent studies have identified several miRNAs in S. mediterranea, their expression in neoblast subpopulations and during regeneration has not been examined. Here, we identify several miRNAs whose expression is enriched in different neoblast subpopulations and in regenerating tissue at different time points in S. mediterranea. Some of these miRNAs were enriched within 3 h post-amputation and may, therefore, play a role in wound healing and/or neoblast migration. Our results also revealed miRNAs, such as sme-miR-2d-3p and the sme-miR-124 family, whose expression is enriched in the cephalic ganglia, are also expressed in the brain primordium during CNS regeneration. These results provide new insight into the potential biological functions of miRNAs in neoblasts and regeneration in planarians. PMID:23974438

Evaluation of Strains and Thicknesses of Pipe Elbows on the Basis of Expressions Resulting from the Eudirective for the Case of Large and Small Deformations

NASA Astrophysics Data System (ADS)

Śloderbach, Z.

2017-12-01

The relations to calculate the maximum value of strains in processes of bending tubes on benders, in stretched layers of tubes, are presented in this work on the basis of the EU-Directive concerning production of pressure equipment. It has been shown that for large deformations that occur during bending of the pipes on knees, logarithmic strain measures (real) and relative strain measures give different values of strain but equal wall thicknesses in the bending zone. Logarithmic measures are frequently used in engineering practice and are valid for large and small deformations. Reverse expressions were also derived to calculate the required initial wall thickness of the tube to be bent, in order to obtain the desired wall thickness of the knee after bending.

Passenger strand loading in overexpression experiments using microRNA mimics.

PubMed

Søkilde, Rolf; Newie, Inga; Persson, Helena; Borg, Åke; Rovira, Carlos

2015-01-01

MicroRNAs (miRNAs) are important regulators of gene function and manipulation of miRNAs is a central component of basic research. Modulation of gene expression by miRNA gain-of-function can be based on different approaches including transfection with miRNA mimics; artificial, chemically modified miRNA-like small RNAs. These molecules are intended to mimic the function of a miRNA guide strand while bypassing the maturation steps of endogenous miRNAs. Due to easy accessibility through commercial providers this approach has gained popularity, and accuracy is often assumed without prior independent testing. Our in silico analysis of over-represented sequence motifs in microarray expression data and sequencing of AGO-associated small RNAs indicate, however, that miRNA mimics may be associated with considerable side-effects due to the unwanted activity of the miRNA mimic complementary strand.

Expression of mucins in the mucosal surface of small intestines in 1 week-old pigs.

PubMed

Kim, Chung Hyun; Oh, Yeonsu; Ha, Yooncheol; Ahn, Qwein; Kim, Sung-Hoon; Cho, Kyung-Dong; Lee, Bog-Hieu; Chae, Chanhee

2010-02-01

The aim of this study was to determine the immunoexpression of mucins in jejunal and ileal villous epithelium using six antibodies against MUC1, MUC2, MUC4 MUC5AC, MUC5B and MUC6. The immunohistochemical score for MUC1 has significantly intense staining compared with MUC2 (P=0.008) and the immunohistochemical socre for MUC4 and MUC 6 has significantly intense staining compared with MUC2 (P=0.032) in ileal villous surface. The immunohistochemical score for MUC4 (P=0.008), MUC5AC (P=0.016) and MUC6 (P=0.016) in ileal villous surface has significantly intense staining compared with ileal cryptic surface. The results of this study demonstrated that six mucins gave distinctly different expression patterns throughout the 1 week-old porcine small intestinal tract.

[A new strategy of gene therapy for hyperphenylalaninemia rats].

PubMed

Jia, X; Liu, J; Xiang, H

2000-06-01

To construct a recombinant vector which expresses active phenylalanine-amonia-lyase (PAL) in Lactococcus lactis (L. L.), and to convert phe into cinnamic acid in small intestine by the engineering L. L. to decrease the phe level in the peripheral blood, and to cure hyperphenylalaninemia rats. PAL cDNA from Petroselinum crispum was subcloned into expression vector pMG36e and transformed L. L. The pMG36ePAL/L.L. was screened and characterized by using PCR and HPLC, and prepared as enteric-coated microcapsules and oral liquid type preparation that were given orally to hyperphenylalaninemia-rats. Engineering L. L. expressing PAL activity was obtained. The phe levels plasma of in the rats receiving preparations made from the engineering L. L. were significantly reduced compared with non-treated hyperphenylalaninemia rats. And the effects of different preparations were different from each other. The engineering L. L. expressing PAL activity can reduce the blood phe level of the hyperphenylalaninemia rats. This may be a potential way for PKU gene therapeutics.

Transcriptomic insights into the genetic basis of mammalian limb diversity.

PubMed

Maier, Jennifer A; Rivas-Astroza, Marcelo; Deng, Jenny; Dowling, Anna; Oboikovitz, Paige; Cao, Xiaoyi; Behringer, Richard R; Cretekos, Chris J; Rasweiler, John J; Zhong, Sheng; Sears, Karen E

2017-03-23

From bat wings to whale flippers, limb diversification has been crucial to the evolutionary success of mammals. We performed the first transcriptome-wide study of limb development in multiple species to explore the hypothesis that mammalian limb diversification has proceeded through the differential expression of conserved shared genes, rather than by major changes to limb patterning. Specifically, we investigated the manner in which the expression of shared genes has evolved within and among mammalian species. We assembled and compared transcriptomes of bat, mouse, opossum, and pig fore- and hind limbs at the ridge, bud, and paddle stages of development. Results suggest that gene expression patterns exhibit larger variation among species during later than earlier stages of limb development, while within species results are more mixed. Consistent with the former, results also suggest that genes expressed at later developmental stages tend to have a younger evolutionary age than genes expressed at earlier stages. A suite of key limb-patterning genes was identified as being differentially expressed among the homologous limbs of all species. However, only a small subset of shared genes is differentially expressed in the fore- and hind limbs of all examined species. Similarly, a small subset of shared genes is differentially expressed within the fore- and hind limb of a single species and among the forelimbs of different species. Taken together, results of this study do not support the existence of a phylotypic period of limb development ending at chondrogenesis, but do support the hypothesis that the hierarchical nature of development translates into increasing variation among species as development progresses.

Gene expression of placental hormones regulating energy balance in small for gestational age neonates.

PubMed

Struwe, Ellen; Berzl, Gabriele M; Schild, Ralf L; Dötsch, Jörg

2009-01-01

Fetal growth restriction is associated with an increased risk for metabolic and cardiovascular disease in later life. To further elucidate mechanisms that might be involved in the process of prenatal programming, we measured the adipokines leptin, resistin, and adiponectin and the GH-releasing hormone ghrelin in the placenta of small for gestational age (SGA) neonates. The control group included 24 placentas of appropriate for gestational age (AGA) newborns, in the study group were 16 placentas of SGA neonates. Gene expression of leptin, resistin, adiponectin, and ghrelin was examined. For hormones showing alterations in gene regulation placental protein expression was measured by Western blot. Placental mRNA expression of leptin was significantly increased in SGA placentas (p=0.0035, related to beta-actin). Protein concentration was increased, as well. There were no differences in placental resistin, adiponectin, or ghrelin gene expressions between SGA neonates and controls. Leptin was the only hormone to demonstrate a significant inverse correlation with birth weight (r=-0.44, p=0.01). Adiponectin correlated significantly with leptin (r=0.53, p=0.0023) and ghrelin (r=0.50, p=0.0045). Placental leptin gene expression and protein concentration showed the expected increase in the SGA group. Leptin was inversely correlated with birth weight. Positive correlation of adiponectin with leptin and ghrelin expression suggests an interaction between these hormones in the placenta. However, the unchanged expression of resistin, adiponectin, and ghrelin in SGA placentas and the absence of correlation with birth weight cast doubt whether these hormones produced in the placenta play a key role in fetal programming.

B Lymphocyte intestinal homing in inflammatory bowel disease

PubMed Central

2011-01-01

Background Inflammatory bowel disease (IBD) is thought to be due to an abnormal interaction between the host immune system and commensal microflora. Within the intestinal immune system, B cells produce physiologically natural antibodies but pathologically atypical anti-neutrophil antibodies (xANCAs) are frequently observed in patients with IBD. The objective is to investigate the localisation of immunoglobulin-producing cells (IPCs) in samples of inflamed intestinal tissue taken from patients with IBD, and their possible relationship with clinical features. Methods The IPCs in small intestinal, colonic and rectal biopsy specimens of patients with IBD were analysed by means of immunofluorescence using polyclonal rabbit anti-human Ig and goat anti-human IgM. The B cell phenotype of the IPC-positive samples was assessed using monoclonal antibodies specific for CD79, CD20, CD23, CD21, CD5, λ and κ chains. Statistical correlations were sought between the histological findings and clinical expression. Results The study involved 96 patients (64 with ulcerative colitis and 32 with Crohn's disease). Two different patterns of B lymphocyte infiltrates were found in the intestinal tissue: one was characterised by a strong to moderate stromal localisation of small IgM+/CD79+/CD20-/CD21-/CD23-/CD5± IPCs (42.7% of cases); in the other (57.3%) no such small IPCs were detected in stromal or epithelial tissues. IPCs were significantly less frequent in the patients with Crohn's disease than in those with ulcerative colitis (p = 0.004). Conclusion Our findings suggest that different immunopathogenetic pathways underlie chronic intestinal inflammation with different clinical expressions. The presence of small B lymphocytes resembling B-1 cells also seemed to be negatively associated with Crohn's disease. It can therefore be inferred that the gut contains an alternative population of B cells that have a regulatory function. PMID:22208453

High p-Smad2 expression in stromal fibroblasts predicts poor survival in patients with clinical stage I to IIIA non-small cell lung cancer.

PubMed

Chen, Yongbing; Xing, Pengfei; Chen, Yuanyuan; Zou, Li; Zhang, Yongsheng; Li, Feng; Lu, Xueguan

2014-11-05

Increasing evidence indicates that the TGFβ/Smad signaling pathway plays a prominent role in tumor initiation, progression, and metastasis. Therefore, we investigate the expression of p-Smad2 in surgical resection specimens from non-small cell lung cancer, and evaluate the prognostic significance of p-Smad2 expression in stromal fibroblasts and cancer cells for patients with clinical stage I to IIIA non-small cell lung cancer. The immunohistochemical expression of p-Smad2 was evaluated in 78 formalin-fixed paraffin-embedded surgical resection specimens from clinical stage I to IIIA non-small cell lung cancer. Correlations between p-Smad2 expression and clinicopathologic characteristics were determined by Chi-square test. The prognostic significance of p-Smad2 expression in stromal fibroblasts and cancer cells with regard to overall survival was determined by Kaplan-Meier. There were 38.5% (30/78) and 92.3% (72/78) patients with high p-Smad2 expression in stromal fibroblasts and cancer cells, respectively. There was a positive correlation between the p-Smad2 expression level in stromal fibroblasts and the p-Smad2 expression level in cancer cells (χ2=4.176, P=0.045). No significant correlation of p-Smad2 expression in stromal fibroblasts or cancer cells with any of clinicopathologic characteristics was found. The 3-year overall survival rates with low and high p-Smad2 expression in stromal fibroblasts were 53.7% and 37.7%, respectively (χ2=3.86, P=0.049). No significant association was found between low and high p-Smad2 expression in cancer cells with respect to overall survival, respectively (χ2=0.34, P=0.562). The results suggested that high p-Smad2 expression in stromal fibroblasts predicted poor survival in patients with clinical stage I to IIIA non-small cell lung cancer.

Insight into small RNA abundance and expression in high- and low-temperature stress response using deep sequencing in Arabidopsis.

PubMed

Baev, Vesselin; Milev, Ivan; Naydenov, Mladen; Vachev, Tihomir; Apostolova, Elena; Mehterov, Nikolay; Gozmanva, Mariyana; Minkov, Georgi; Sablok, Gaurav; Yahubyan, Galina

2014-11-01

Small RNA profiling and assessing its dependence on changing environmental factors have expanded our understanding of the transcriptional and post-transcriptional regulation of plant stress responses. Insufficient data have been documented earlier to depict the profiling of small RNA classes in temperature-associated stress which has a wide implication for climate change biology. In the present study, we report a comparative assessment of the genome-wide profiling of small RNAs in Arabidopsis thaliana using two conditional responses, induced by high- and low-temperature. Genome-wide profiling of small RNAs revealed an abundance of 21 nt small RNAs at low temperature, while high temperature showed an abundance of 21 nt and 24 nt small RNAs. The two temperature treatments altered the expression of a specific subset of mature miRNAs and displayed differential expression of a number of miRNA isoforms (isomiRs). Comparative analysis demonstrated that a large number of protein-coding genes can give rise to differentially expressed small RNAs following temperature shifts. Low temperature caused accumulation of small RNAs, corresponding to the sense strand of a number of cold-responsive genes. In contrast, high temperature stimulated the production of small RNAs of both polarities from genes encoding functionally diverse proteins. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

The miRNA Transcriptome Directly Reflects the Physiological and Biochemical Differences between Red, White, and Intermediate Muscle Fiber Types.

PubMed

Ma, Jideng; Wang, Hongmei; Liu, Rui; Jin, Long; Tang, Qianzi; Wang, Xun; Jiang, Anan; Hu, Yaodong; Li, Zongwen; Zhu, Li; Li, Ruiqiang; Li, Mingzhou; Li, Xuewei

2015-04-29

MicroRNAs (miRNAs) are small non-coding RNAs that can regulate their target genes at the post-transcriptional level. Skeletal muscle comprises different fiber types that can be broadly classified as red, intermediate, and white. Recently, a set of miRNAs was found expressed in a fiber type-specific manner in red and white fiber types. However, an in-depth analysis of the miRNA transcriptome differences between all three fiber types has not been undertaken. Herein, we collected 15 porcine skeletal muscles from different anatomical locations, which were then clearly divided into red, white, and intermediate fiber type based on the ratios of myosin heavy chain isoforms. We further illustrated that three muscles, which typically represented each muscle fiber type (i.e., red: peroneal longus (PL), intermediate: psoas major muscle (PMM), white: longissimus dorsi muscle (LDM)), have distinct metabolic patterns of mitochondrial and glycolytic enzyme levels. Furthermore, we constructed small RNA libraries for PL, PMM, and LDM using a deep sequencing approach. Results showed that the differentially expressed miRNAs were mainly enriched in PL and played a vital role in myogenesis and energy metabolism. Overall, this comprehensive analysis will contribute to a better understanding of the miRNA regulatory mechanism that achieves the phenotypic diversity of skeletal muscles.

Gender Difference of Hepatic and Intestinal CYP3A4 in CYP3AHumanized Mice Generated by a Human Chromosome-engineering Technique.

PubMed

Kobayashi, Kaoru; Abe, Chihiro; Endo, Mika; Kazuki, Yasuhiro; Oshimura, Mitsuo; Chiba, Kan

2017-11-17

Cytochrome P450 3A4 (CYP3A4) is an important drug-metabolizing enzyme that is expressed in the liver and small intestine of humans. Various factors influence the expression of CYP3A4, but gender difference in CYP3A4 expression remains debatable. To clarify gender difference of hepatic and intestinal CYP3A4 in CYP3A-humanized mice generated by a human artificial chromosome (HAC) vector system. The CYP3A-humanized (CYP3AHAC) mice have essential regulatory regions, including promoters and enhancers, and unknown elements affecting the expression of CYP3A4. We examined the expression and activity of hepatic and intestinal CYP3A4 in male and female CYP3A-HAC mice. CYP3A activity was determined as α- and 4-hydroxylation activity of triazolam in liver and intestinal microsomes. Expression level of CYP3A protein was determined by Western blot analysis. Expression level of CYP3A4 mRNA was measured by quantitative real-time PCR. The results showed that triazolam hydroxylation activities and protein levels of CYP3A in the liver were significantly higher in female than in male CYP3A-HAC mice, whereas those in the intestine were not significantly different between the genders. In addition, the expression of CYP3A4 mRNA showed a tendency similar to that found for the activity and expression of CYP3A protein in the liver and intestine of CYP3A-HAC mice. These findings suggest that the expression and activity levels of CYP3A4 in the liver are higher in females than in males, whereas there is no gender difference in the levels in the intestine of CYP3A-HAC mice. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Quality control in molecular immunohistochemistry

PubMed Central

2008-01-01

Immunoperoxidase histochemistry is a widespread method of assessing expression of biomolecules in tissue samples. Accurate assessment of the expression levels of genes is critical for the management of disease, particularly as therapy targeted to specific molecules becomes more widespread. Determining the quality of preservation of macromolecules in tissue is important to avoid false negative and false positive results. In this review we discuss (1) issues of sensitivity (false negativity) and specificity (false positivity) of immunohistochemical stains, (2) approaches to better understanding differences in immunostains done by different laboratories (including the recently proposed MISFISHIE specification for tissue localization studies), and (3) approaches to assessing the quality of preservation of macromolecules in tissue, particularly in small biopsy samples. PMID:18648842

The Expression of NOX4 in Smooth Muscles of Small Airway Correlates with the Disease Severity of COPD

PubMed Central

2016-01-01

Airway smooth muscle (ASM) remodeling is a hallmark in chronic obstructive pulmonary disease (COPD), and nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases (NOXs) produced reactive oxygen species (ROS) play a crucial role in COPD pathogenesis. In the present study, the expression of NOX4 and its correlation with the ASM hypertrophy/hyperplasia, clinical pulmonary functions, and the expression of transforming growth factor β (TGF-β) in the ASM of COPD small airways were investigated by semiquantitative morphological and/or immunohistochemistry staining methods. The results showed that an elevated expression of NOX4 and TGF-β, along with an increased volume of ASM mass, was found in the ASM of small airways in COPD patients. The abundance of NOX4 protein in the ASM was increased with disease severity and inversely correlated with the pulmonary functions in COPD patients. In addition, the expression of NOX4 and ASM marker α-SMA was colocalized, and the increased NOX4 expression was found to accompany an upregulated expression of TGF-β in the ASM of small airways of COPD lung. These results indicate that NOX4 may be a key regulator in ASM remodeling of small airway, in part through a mechanism interacting with TGF-β signaling in the pathogenesis of COPD, which warrants further investigation. PMID:27656649

Comparison of small biopsy specimens and surgical specimens for the detection of EGFR mutations and EML4-ALK in non-small-cell lung cancer

PubMed Central

Xiao, DeSheng; Lu, Can; Zhu, Wei; He, QiuYan; Li, Yong; Fu, ChunYan; Zhou, JianHua; Liu, Shuang; Tao, YongGuang

2016-01-01

Epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) fusion genes represent novel oncogenes that are associated with non–small-cell lung cancers (NSCLC). The feasibility of detecting EGFR mutations and ALK fusion genes in small biopsy specimens or surgical specimens was determined. Of the 721 NSCLC patients, a total of 305 cases were positive for EGFR mutations (42.3%). The rate of EGFR mutations in women was significantly higher than that in men. Histologically, the EGFR mutation rate in adenocarcinomas was significantly higher than that in squamous cell carcinomas. No difference in the EGFR mutation rate was observed between surgical specimens (42.1%) and small biopsy specimens (42.4%), which indicated that the EGFR mutation ratios in surgical specimens and small biopsy specimens were not different. In 385 NSCLC patients, 26 cases were positive for EML4-ALK (6.8%). However, 11.7% of the surgical specimens were EML4-ALK-positive, whereas the positive proportion in the small biopsy specimens was only 4.7%, which indicated that EML4-ALK-positive rate in the surgical specimens was significantly higher than that in the small biopsy specimens. Detection of EGFR gene mutations was feasible in small biopsy specimens, and screening for EML4-ALK expression in small biopsy specimens can be used to guide clinical treatments. PMID:27322143

Comparison of small biopsy specimens and surgical specimens for the detection of EGFR mutations and EML4-ALK in non-small-cell lung cancer.

PubMed

Xiao, DeSheng; Lu, Can; Zhu, Wei; He, QiuYan; Li, Yong; Fu, ChunYan; Zhou, JianHua; Liu, Shuang; Tao, YongGuang

2016-09-13

Epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) fusion genes represent novel oncogenes that are associated with non-small-cell lung cancers (NSCLC). The feasibility of detecting EGFR mutations and ALK fusion genes in small biopsy specimens or surgical specimens was determined. Of the 721 NSCLC patients, a total of 305 cases were positive for EGFR mutations (42.3%). The rate of EGFR mutations in women was significantly higher than that in men. Histologically, the EGFR mutation rate in adenocarcinomas was significantly higher than that in squamous cell carcinomas. No difference in the EGFR mutation rate was observed between surgical specimens (42.1%) and small biopsy specimens (42.4%), which indicated that the EGFR mutation ratios in surgical specimens and small biopsy specimens were not different. In 385 NSCLC patients, 26 cases were positive for EML4-ALK (6.8%). However, 11.7% of the surgical specimens were EML4-ALK-positive, whereas the positive proportion in the small biopsy specimens was only 4.7%, which indicated that EML4-ALK-positive rate in the surgical specimens was significantly higher than that in the small biopsy specimens. Detection of EGFR gene mutations was feasible in small biopsy specimens, and screening for EML4-ALK expression in small biopsy specimens can be used to guide clinical treatments.

KRAS-mutation status dependent effect of zoledronic acid in human non-small cell cancer preclinical models

PubMed Central

Kenessey, István; Kói, Krisztina; Horváth, Orsolya; Cserepes, Mihály; Molnár, Dávid; Izsák, Vera; Dobos, Judit; Hegedűs, Balázs

2016-01-01

Background In non-small cell lung cancer (NSCLC) KRAS-mutant status is a negative prognostic and predictive factor. Nitrogen-containing bisphosphonates inhibit prenylation of small G-proteins (e.g. Ras, Rac, Rho) and thus may affect proliferation and migration. In our preclinical work, we investigated the effect of an aminobisphosphonate compound (zoledronic acid) on mutant and wild type KRAS-expressing human NSCLC cell lines. Results We confirmed that zoledronic acid was unable to inhibit the prenylation of mutant K-Ras unlike in the case of wild type K-Ras. In case of in vitro proliferation, the KRAS-mutant human NSCLC cell lines showed resistance to zoledronic acid wild-type KRAS-cells proved to be sensitive. Combinatory application of zoledronic acid enhanced the cytostatic effect of cisplatin. Zoledronic acid did not induce significant apoptosis. In xenograft model, zoledronic acid significantly reduced the weight of wild type KRAS-EGFR-expressing xenograft tumor by decreasing the proliferative capacity. Futhermore, zoledronic acid induced VEGF expression and improved in vivo tumor vascularization. Materials and methods Membrane association of K-Ras was examined by Western-blot. In vitro cell viability, apoptotic cell death and migration were measured in NSCLC lines with different molecular background. The in vivo effect of zoledronic acid was investigated in a SCID mouse subcutaneous xenograft model. Conclusions The in vitro and in vivo inhibitory effect of zoledronic acid was based on the blockade of cell cycle in wild type KRAS-expressing human NSCLC cells. The zoledronic acid induced vascularization supported in vivo cytostatic effect. Our preclinical investigation suggests that patients with wild type KRAS-expressing NSCLC could potentially benefit from aminobisphosphonate therapy. PMID:27780929

Subcellular localization and distribution of the reduced folate carrier in normal rat tissues.

PubMed

Hinken, M; Halwachs, S; Kneuer, C; Honscha, W

2011-01-27

The reduced folate carrier (Rfc1; Slc19a1) mediated transport of reduced folates and antifolate drugs such as methotrexate (MTX) play an essential role in physiological folate homeostasis and MTX cancer chemotherapy. As no systematic reports are as yet available correlating Rfc1 gene expression and protein levels in all tissues crucial for folate and antifolate uptake, storage or elimination, we investigated gene and protein expression of rat Rfc1 (rRfc1) in selected tissues. This included the generation of a specific anti-rRfc1 antibody. Rabbits were immunised with isolated rRfc1 peptides producing specific anti-rRfc1 antiserum targeted to the intracellular C-terminus of the carrier. Using RT-PCR analysis, high rRfc1 transcript levels were detected in colon, kidney, brain, thymus, and spleen. Moderate rRfc1 gene expression was observed in small intestine, liver, bone marrow, lung, and testes whereas transcript levels were negligible in heart, skeletal muscle or leukocytes. Immunohistochemical analyses revealed strong carrier expression in the apical membrane of tunica mucosa epithelial cells of small intestine and colon, in the brush-border membrane of choroid plexus epithelial cells or in endothelial cells of small vessels in brain and heart. Additionally, high rRfc1 protein levels were localized in the basolateral membrane of renal tubular epithelial cells, in the plasma membrane of periportal hepatocytes, and sertoli cells of the testes. Taken together, our results demonstrated that rRfc1 is expressed almost ubiquitously but to very different levels. The predominant tissue distribution supports the essential role of Rfc1 in physiological folate homeostasis. Moreover, our results may contribute to understand antifolate pharmacokinetics and selected organ toxicity associated with MTX chemotherapy.

Non-Small-Cell Lung Cancer Molecular Signatures Recapitulate Lung Developmental Pathways

PubMed Central

Borczuk, Alain C.; Gorenstein, Lyall; Walter, Kristin L.; Assaad, Adel A.; Wang, Liqun; Powell, Charles A.

2003-01-01

Current paradigms hold that lung carcinomas arise from pleuripotent stem cells capable of differentiation into one or several histological types. These paradigms suggest lung tumor cell ontogeny is determined by consequences of gene expression that recapitulate events important in embryonic lung development. Using oligonucleotide microarrays, we acquired gene profiles from 32 microdissected non-small-cell lung tumors. We determined the 100 top-ranked marker genes for adenocarcinoma, squamous cell, large cell, and carcinoid using nearest neighbor analysis. Results were validated by immunostaining for 11 selected proteins using a tissue microarray representing 80 tumors. Gene expression data of lung development were accessed from a publicly available dataset generated with the murine Mu11k genome microarray. Self-organized mapping identified two temporally distinct clusters of murine orthologues. Supervised clustering of lung development data showed large-cell carcinoma gene orthologues were in a cluster expressed in pseudoglandular and canalicular stages whereas adenocarcinoma homologues were predominantly in a cluster expressed later in the terminal sac and alveolar stages of murine lung development. Representative large-cell genes (E2F3, MYBL2, HDAC2, CDK4, PCNA) are expressed in the nucleus and are associated with cell cycle and proliferation. In contrast, adenocarcinoma genes are associated with lung-specific transcription pathways (SFTPB, TTF-1), cell adhesion, and signal transduction. In sum, non-small-cell lung tumors histology gene profiles suggest mechanisms relevant to ontogeny and clinical course. Adenocarcinoma genes are associated with differentiation and glandular formation whereas large-cell genes are associated with proliferation and differentiation arrest. The identification of developmentally regulated pathways active in tumorigenesis provides insights into lung carcinogenesis and suggests early steps may differ according to the eventual tumor morphology. PMID:14578194

Differences in cumulus cells gene expression between modified natural and stimulated in vitro fertilization cycles.

PubMed

Papler, Tanja Burnik; Bokal, Eda Vrtačnik; Tacer, Klementina Fon; Juvan, Peter; Virant Klun, Irma; Devjak, Rok

2014-01-01

The aim of our study was to determine whether there are any differences in the cumulus cell gene expression profile of mature oocytes derived from modified natural IVF and controlled ovarian hyperstimulation cycles and if these changes could help us understand why modified natural IVF has lower success rates. Cumulus cells surrounding mature oocytes that developed to morulae or blastocysts on day 5 after oocyte retrieval were submitted to microarray analysis. The obtained data were then validated using quantitative real-time PCR. There were 66 differentially expressed genes between cumulus cells of modified natural IVF and controlled ovarian hyperstimulation cycles. Gene ontology analysis revealed the oxidation-reduction process, glutathione metabolic process, xenobiotic metabolic process and gene expression were significantly enriched biological processes in MNIVF cycles. Among differentially expressed genes we observed a large group of small nucleolar RNA's whose role in folliculogenesis has not yet been established. The increased expression of genes involved in the oxidation-reduction process probably points to hypoxic conditions in modified natural IVF cycles. This finding opens up new perspectives for the establishment of the potential role that oxidation-reduction processes have in determining success rates of modified natural IVF.

RNA-based regulation of genes of tryptophan synthesis and degradation, in bacteria

PubMed Central

Yanofsky, Charles

2007-01-01

We are now aware that RNA-based regulatory mechanisms are commonly used to control gene expression in many organisms. These mechanisms offer the opportunity to exploit relatively short, unique RNA sequences, in altering transcription, translation, and/or mRNA stability, in response to the presence of a small or large signal molecule. The ability of an RNA segment to fold and form alternative hairpin secondary structures—each dedicated to a different regulatory function—permits selection of specific sequences that can affect transcription and/or translation. In the present paper I will focus on our current understanding of the RNA-based regulatory mechanisms used by Escherichia coli and Bacillus subtilis in controlling expression of the tryptophan biosynthetic operon. The regulatory mechanisms they use for this purpose differ, suggesting that these organisms, or their ancestors, adopted different strategies during their evolution. I will also describe the RNA-based mechanism used by E. coli in regulating expression of its operon responsible for tryptophan degradation, the tryptophanase operon. PMID:17601995

Distinct TrkA and Ret modulated negative and positive neuropathic behaviors in a mouse model of resiniferatoxin-induced small fiber neuropathy.

PubMed

Hsieh, Yu-Lin; Kan, Hung-Wei; Chiang, Hao; Lee, Yi-Chen; Hsieh, Sung-Tsang

2018-02-01

Neurotrophic factors and their corresponding receptors play key roles in the maintenance of different phenotypic dorsal root ganglion (DRG) neurons, the axons of which degenerate in small fiber neuropathy, leading to various neuropathic manifestations. Mechanisms underlying positive and negative symptoms of small fiber neuropathy have not been systematically explored. This study investigated the molecular basis of these seemingly paradoxical neuropathic behaviors according to the profiles of TrkA and Ret with immunohistochemical and pharmacological interventions in a mouse model of resiniferatoxin (RTX)-induced small fiber neuropathy. Mice with RTX neuropathy exhibited thermal hypoalgesia and mechanical allodynia, reduced skin innervation, and altered DRG expression profiles with decreased TrkA(+) neurons and increased Ret(+) neurons. RTX neuropathy induced the expression of activating transcription factor 3 (ATF3), and ATF3(+) neurons were colocalized with Ret but not with TrkA (P<0.001). As a neuroprotectant, 4-Methylcatechol (4MC) promoted skin reinnervation partially with correlated reversal of the neuropathic behaviors and altered neurochemical expression. Gambogic amide, a selective TrkA agonist, normalized thermal hypoalgesia, and GW441756, a TrkA kinase inhibitor, induced thermal hypoalgesia, which was already reversed by 4MC. Mechanical allodynia was reversed by a Ret kinase inhibitor, AST487, which induced thermal hyperalgesia in naïve mice. The activation of Ret signaling by XIB4035 induced mechanical allodynia and thermal hypoalgesia in RTX neuropathy mice in which the neuropathic behaviors were previously normalized by 4MC. Distinct neurotrophic factor receptors, TrkA and Ret, accounted for negative and positive neuropathic behaviors in RTX-induced small fiber neuropathy, respectively: TrkA for thermal hypoalgesia and Ret for mechanical allodynia and thermal hypoalgesia. Copyright © 2017 Elsevier Inc. All rights reserved.

Unraveling Regulation of the Small Heat Shock Proteins by the Heat Shock Factor HvHsfB2c in Barley: Its Implications in Drought Stress Response and Seed Development

PubMed Central

Reddy, Palakolanu Sudhakar; Kavi Kishor, Polavarapu B.; Seiler, Christiane; Kuhlmann, Markus; Eschen-Lippold, Lennart; Lee, Justin; Reddy, Malireddy K.; Sreenivasulu, Nese

2014-01-01

The rapid increase in heat shock proteins upon exposure to damaging stresses and during plant development related to desiccation events reveal their dual importance in plant development and stress tolerance. Genome-wide sequence survey identified 20 non-redundant small heat shock proteins (sHsp) and 22 heat shock factor (Hsf) genes in barley. While all three major classes (A, B, C) of Hsfs are localized in nucleus, the 20 sHsp gene family members are localized in different cell organelles like cytoplasm, mitochondria, plastid and peroxisomes. Hsf and sHsp members are differentially regulated during drought and at different seed developmental stages suggesting the importance of chaperone role under drought as well as seed development. In silico cis-regulatory motif analysis of Hsf promoters showed an enrichment with abscisic acid responsive cis-elements (ABRE), implying regulatory role of ABA in mediating transcriptional response of HvsHsf genes. Gene regulatory network analysis identified HvHsfB2c as potential central regulator of the seed-specific expression of several HvsHsps including 17.5CI sHsp. These results indicate that HvHsfB2c is co-expressed in the central hub of small Hsps and therefore it may be regulating the expression of several HvsHsp subclasses HvHsp16.88-CI, HvHsp17.5-CI and HvHsp17.7-CI. The in vivo relevance of binding specificity of HvHsfB2C transcription factor to HSE-element present in the promoter of HvSHP17.5-CI under heat stress exposure is confirmed by gel shift and LUC-reporter assays. Further, we isolated 477 bp cDNA from barley encoding a 17.5 sHsp polypeptide, which was predominantly upregulated under drought stress treatments and also preferentially expressed in developing seeds. Recombinant HvsHsp17.5-CI protein was expressed in E. coli and purified to homogeneity, which displayed in vitro chaperone activity. The predicted structural model of HvsHsp-17.5-CI protein suggests that the α-crystallin domain is evolutionarily highly conserved. PMID:24594978

Unraveling regulation of the small heat shock proteins by the heat shock factor HvHsfB2c in barley: its implications in drought stress response and seed development.

PubMed

Reddy, Palakolanu Sudhakar; Kavi Kishor, Polavarapu B; Seiler, Christiane; Kuhlmann, Markus; Eschen-Lippold, Lennart; Lee, Justin; Reddy, Malireddy K; Sreenivasulu, Nese

2014-01-01

The rapid increase in heat shock proteins upon exposure to damaging stresses and during plant development related to desiccation events reveal their dual importance in plant development and stress tolerance. Genome-wide sequence survey identified 20 non-redundant small heat shock proteins (sHsp) and 22 heat shock factor (Hsf) genes in barley. While all three major classes (A, B, C) of Hsfs are localized in nucleus, the 20 sHsp gene family members are localized in different cell organelles like cytoplasm, mitochondria, plastid and peroxisomes. Hsf and sHsp members are differentially regulated during drought and at different seed developmental stages suggesting the importance of chaperone role under drought as well as seed development. In silico cis-regulatory motif analysis of Hsf promoters showed an enrichment with abscisic acid responsive cis-elements (ABRE), implying regulatory role of ABA in mediating transcriptional response of HvsHsf genes. Gene regulatory network analysis identified HvHsfB2c as potential central regulator of the seed-specific expression of several HvsHsps including 17.5CI sHsp. These results indicate that HvHsfB2c is co-expressed in the central hub of small Hsps and therefore it may be regulating the expression of several HvsHsp subclasses HvHsp16.88-CI, HvHsp17.5-CI and HvHsp17.7-CI. The in vivo relevance of binding specificity of HvHsfB2C transcription factor to HSE-element present in the promoter of HvSHP17.5-CI under heat stress exposure is confirmed by gel shift and LUC-reporter assays. Further, we isolated 477 bp cDNA from barley encoding a 17.5 sHsp polypeptide, which was predominantly upregulated under drought stress treatments and also preferentially expressed in developing seeds. Recombinant HvsHsp17.5-CI protein was expressed in E. coli and purified to homogeneity, which displayed in vitro chaperone activity. The predicted structural model of HvsHsp-17.5-CI protein suggests that the α-crystallin domain is evolutionarily highly conserved.

Flow cytometric analysis of immunoglobulin heavy chain expression in B-cell lymphoma and reactive lymphoid hyperplasia

PubMed Central

Grier, David D; Al-Quran, Samer Z; Cardona, Diana M; Li, Ying; Braylan, Raul C

2012-01-01

The diagnosis of B-cell lymphoma (BCL) is often dependent on the detection of clonal immunoglobulin (Ig) light chain expression. In some BCLs, the determination of clonality based on Ig light chain restriction may be difficult. The aim of our study was to assess the utility of flow cytometric analysis of surface Ig heavy chain (HC) expression in lymphoid tissues in distinguishing lymphoid hyperplasias from BCLs, and also differentiating various BCL subtypes. HC expression on B-cells varied among different types of hyperplasias. In follicular hyperplasia, IgM and IgD expression was high in mantle cells while germinal center cells showed poor HC expression. In other hyperplasias, B cell compartments were blurred but generally showed high IgD and IgM expression. Compared to hyperplasias, BCLs varied in IgM expression. Small lymphocytic lymphomas had lower IgM expression than mantle cell lymphomas. Of importance, IgD expression was significantly lower in BCLs than in hyperplasias, a finding that can be useful in differentiating lymphoma from reactive processes. PMID:22400070

In vitro synergistic antitumor efficacy of sequentially combined chemotherapy/icotinib in non‑small cell lung cancer cell lines.

PubMed

Wang, Min-Cong; Liang, Xuan; Liu, Zhi-Yan; Cui, Jie; Liu, Ying; Jing, Li; Jiang, Li-Li; Ma, Jie-Qun; Han, Li-Li; Guo, Qian-Qian; Yang, Cheng-Cheng; Wang, Jing; Wu, Tao; Nan, Ke-Jun; Yao, Yu

2015-01-01

The concurrent administration of chemotherapy and epidermal growth factor receptor‑tyrosine kinase inhibitors (EGFR‑TKIs) has previously produced a negative interaction and failed to confer a survival benefit to non‑small cell lung cancer (NSCLC) patients compared with first‑line cytotoxic chemotherapy. The present study aimed to investigate the optimal schedule of the combined treatment of cisplatin/paclitaxel and icotinib in NSCLC cell lines and clarify the underlying mechanisms. HCC827, H1975, H1299 and A549 human NSCLC cell lines with wild‑type and mutant EGFR genes were used as in vitro models to define the differential effects of various schedules of cisplatin/paclitaxel with icotinib treatments on cell growth, proliferation, cell cycle distribution, apoptosis, and EGFR signaling pathway. Sequence‑dependent antiproliferative effects differed among the four NSCLC cell lines, and were not associated with EGFR mutation, constitutive expression levels of EGFR or downstream signaling molecules. The antiproliferative effect of cisplatin plus paclitaxel followed by icotinib was superior to that of cisplatin or paclitaxel followed by icotinib in the HCC827, H1975, H1299 and A549 cell lines, and induced more cell apoptosis and G0/G1 phase arrest. Cisplatin and paclitaxel significantly increased the expression of EGFR phosphorylation in the HCC827 cell line. However, only paclitaxel increased the expression of EGFR phosphorylation in the H1975 cell line. Cisplatin/paclitaxel followed by icotinib influenced the expression of p‑EGFR and p‑AKT, although the expression of p‑ERK1/2 remained unchanged. The results suggest that the optimal schedule of the combined treatment of cisplatin/paclitaxel and icotinib differed among the NSCLC cell lines. The results also provide molecular evidence to support clinical treatment strategies for NSCLC patients.

Expression of PD-1 and PD-L1 in poorly differentiated neuroendocrine carcinomas of the digestive system: a potential target for anti-PD-1/PD-L1 therapy.

PubMed

Roberts, Jordan A; Gonzalez, Raul S; Das, Satya; Berlin, Jordan; Shi, Chanjuan

2017-12-01

Poorly differentiated neuroendocrine carcinoma of the digestive system has a dismal prognosis with limited treatment options. This study aimed to investigate expression of the PD-1/PD-L1 pathway in these tumors. Thirty-seven patients with a poorly differentiated neuroendocrine carcinoma of the digestive system were identified. Their electronic medical records, pathology reports, and pathology slides were reviewed for demographics, clinical history, and pathologic features. Tumor sections were immunohistochemically labeled for PD-1 and PD-L1, and expression of PD-1 and PD-L1 on tumor and tumor-associated immune cells was analyzed and compared between small cell and large cell neuroendocrine carcinomas. The mean age of patients was 61 years old with 18 men and 19 women. The colorectum (n=20) was the most common primary site; other primary sites included the pancreaticobiliary system, esophagus, stomach, duodenum, and ampulla. Expression of PD-1 was detected on tumor cells (n=6, 16%) as well as on tumor-associated immune cells (n=23, 63%). The 6 cases with PD-1 expression on tumor cells also had the expression on immune cells. Expression of PD-L1 was visualized on tumor cells in 5 cases (14%) and on tumor-associated immune cells in 10 cases (27%). There was no difference in PD-1 and PD-L1 expression between small cell and large cell neuroendocrine carcinomas. In conclusion, PD-1/PD-L1 expression is a frequent occurrence in poorly differentiated neuroendocrine carcinomas of the digestive system. Checkpoint blockade targeting the PD-1/PD-L1 pathway may have a potential role in treating patients with this disease. Copyright © 2017 Elsevier Inc. All rights reserved.

[Effects of Losartan on expression of heme oxygenases in volume-overloaded rats with left-to-right shunt].

PubMed

Yuan, Li-Xing; Liu, Han-Min; Li, Mi; Gao, Ju; Zhou, Tong-Fu

2005-09-01

To study the expression of heme oxygenase-1 mRNA and pulmonary remodeling before and after surgical establishment of left-to-right shunt in volume-overloaded SD rats and rats with Losartan intervention. Left-to-right shunt volume-overloaded SD rat models were established by aortocaval shunt operation. Seven rats with shunt were placed on Losartan (Losartan group), 7 rats with but not given Losartan were included in the operation group, and 4 rats after sham operation served as controls. Pulmonary pressure and right ventricular pressure were measured during catheterization. The relative weights ventricles were determined after execution of the rats. Pulmonary vascular remodeling parameters, including percentage arterial wall thickness and percentage muscularized small arteries, were assessed by morphometry. Heme oxygenase-1 (HO-1) mRNA expression and heme oxygenase-2 (HO-2) mRNA expression were detected RT-PCR method. Pulmonary artery pressure and right ventricular relative weight decreased significantly in the rats of Losartan group; in addition, the percentage arterial wall thickness and percentage of muscularized small arteries in the Losartan group were reduced as compared with those in the operation group. The level 1 mRAN expression in rats with shunt was significantly higher than that in rats without shunt. The level mRNA expression in the Losartan group decreased remarkably as compared against that in the operation The level of HO-1 mRNA expression in lungs was significantly higher than that in ventricles. There statistically significant differences in HO-2 mRNA expression levels between the three rat groups. Losartan intervention can markedly reduce pulmonary pressure, inhibit vascular remodeling in volume-overloaded left-to-right shunt rats, and result in down-regulation of HO-1 mRNA expression.

Lung cancer incidence and survival in chromium exposed individuals with respect to expression of anti-apoptotic protein survivin and tumor suppressor P53 protein

PubMed Central

2010-01-01

Objective Workers chronically exposed to hexavalent chromium have elevated risk of lung cancer. Our study investigates the incidence of lung cancer types, age at onset of the disease, and survival time among chromium exposed workers with respect to the expression of anti-apoptotic p53 and pro-apoptotic survivin proteins. Materials and methods 67 chromium exposed workers and 104 male controls diagnosed with lung cancer were analyzed. The mean exposure time among workers was 16.7 ± 10.0(SD) years (range 1- 41 years). To investigate the possible regulation of survivin by p53 we examined the expression of both proteins using immohistochemical visualization. Results Chromium exposure significantly decreases the age of onset of the disease by 3.5 years (62.2 ± 9.1 in the exposed group vs. 65.7 ± 10.5 years in controls; P = 0.018). Small cell lung carcinoma (SCLC) amounted for 25.4% of all cases in chromium exposed workers and for 16.3% in non-exposed individuals. The mean survival time in the exposed group was 9.0 ± 12.7 vs. 12.1 ± 21.9 months in controls, but this difference was not significant. Survivin was predominantly expressed in both cell nucleus and cytoplasm, whereas p53 was expressed in the nucleus. There was a negative correlation between survivin and p53 expression. A decreased intensity of expression and fewer cells positive for survivin was detected in SCLC compared with other types of lung cancer. P53 was expressed in 94.1% and survivin in 79.6% of the samples analyzed. Conclusion The study calls attention to decreased expression of survivin, as opposed to p53, in small cell lung carcinoma. PMID:21147621

RNA-seq reveals distinctive RNA profiles of small extracellular vesicles from different human liver cancer cell lines.

PubMed

Berardocco, Martina; Radeghieri, Annalisa; Busatto, Sara; Gallorini, Marialucia; Raggi, Chiara; Gissi, Clarissa; D'Agnano, Igea; Bergese, Paolo; Felsani, Armando; Berardi, Anna C

2017-10-10

Liver cancer (LC) is one of the most common cancers and represents the third highest cause of cancer-related deaths worldwide. Extracellular vesicle (EVs) cargoes, which are selectively enriched in RNA, offer great promise for the diagnosis, prognosis and treatment of LC. Our study analyzed the RNA cargoes of EVs derived from 4 liver-cancer cell lines: HuH7, Hep3B, HepG2 (hepato-cellular carcinoma) and HuH6 (hepatoblastoma), generating two different sets of sequencing libraries for each. One library was size-selected for small RNAs and the other targeted the whole transcriptome. Here are reported genome wide data of the expression level of coding and non-coding transcripts, microRNAs, isomiRs and snoRNAs providing the first comprehensive overview of the extracellular-vesicle RNA cargo released from LC cell lines. The EV-RNA expression profiles of the four liver cancer cell lines share a similar background, but cell-specific features clearly emerge showing the marked heterogeneity of the EV-cargo among the individual cell lines, evident both for the coding and non-coding RNA species.

The Distributions of Voltage-Gated K+ current Subtypes in Different Cell Sizes from Adult Mouse Dorsal Root Ganglia.

PubMed

Sheng, Anqi; Hong, Jiangru; Zhang, Lulu; Zhang, Yan; Zhang, Guangqin

2018-03-29

Voltage-gated K + (K V ) currents play a crucial role in regulating pain by controlling neuronal excitability, and are divided into transient A-type currents (I A ) and delayed rectifier currents (I K ). The dorsal root ganglion (DRG) neurons are heterogeneous and the subtypes of K V currents display different levels in distinct cell sizes. To observe correlations of the subtypes of K V currents with DRG cell sizes, K V currents were recorded by whole-cell patch clamp in freshly isolated mouse DRG neurons. Results showed that I A occupied a high proportion in K V currents in medium- and large-diameter DRG neurons, whereas I K possessed a larger proportion of K V currents in small-diameter DRG neurons. A lower correlation was found between the proportion of I A or I K in K V currents and cell sizes. These data suggest that I A channels are mainly expressed in medium and large cells and I K channels are predominantly expressed in small cells.

Gene expression patterns in peripheral blood leukocytes in patients with recurrent ciguatera fish poisoning: Preliminary studies.

PubMed

Lopez, Maria-Cecilia; Ungaro, Ricardo F; Baker, Henry V; Moldawer, Lyle L; Robertson, Alison; Abbott, Margaret; Roberts, Sparkle M; Grattan, Lynn M; Morris, J Glenn

2016-07-01

Ciguatera fish poisoning (ciguatera) is a common clinical syndrome in areas where there is dependence on tropical reef fish for food. A subset of patients develops recurrent and, in some instances, chronic symptoms, which may result in substantial disability. To identify possible biomarkers for recurrent/chronic disease, and to explore correlations with immune gene expression, peripheral blood leukocyte gene expression in 10 ciguatera patients (7 recurrent and 3 acute) from the U.S. Virgin Islands, and 5 unexposed Florida controls were evaluated. Significant differences in gene expression were noted when comparing ciguatera patients and controls; however, it was not possible to differentiate between patients with acute and recurrent disease, possibly due to the small sample sizes involved. Copyright © 2016 Elsevier B.V. All rights reserved.

The landscape of genomic imprinting across diverse adult human tissues.

PubMed

Baran, Yael; Subramaniam, Meena; Biton, Anne; Tukiainen, Taru; Tsang, Emily K; Rivas, Manuel A; Pirinen, Matti; Gutierrez-Arcelus, Maria; Smith, Kevin S; Kukurba, Kim R; Zhang, Rui; Eng, Celeste; Torgerson, Dara G; Urbanek, Cydney; Li, Jin Billy; Rodriguez-Santana, Jose R; Burchard, Esteban G; Seibold, Max A; MacArthur, Daniel G; Montgomery, Stephen B; Zaitlen, Noah A; Lappalainen, Tuuli

2015-07-01

Genomic imprinting is an important regulatory mechanism that silences one of the parental copies of a gene. To systematically characterize this phenomenon, we analyze tissue specificity of imprinting from allelic expression data in 1582 primary tissue samples from 178 individuals from the Genotype-Tissue Expression (GTEx) project. We characterize imprinting in 42 genes, including both novel and previously identified genes. Tissue specificity of imprinting is widespread, and gender-specific effects are revealed in a small number of genes in muscle with stronger imprinting in males. IGF2 shows maternal expression in the brain instead of the canonical paternal expression elsewhere. Imprinting appears to have only a subtle impact on tissue-specific expression levels, with genes lacking a systematic expression difference between tissues with imprinted and biallelic expression. In summary, our systematic characterization of imprinting in adult tissues highlights variation in imprinting between genes, individuals, and tissues. © 2015 Baran et al.; Published by Cold Spring Harbor Laboratory Press.

Validating internal controls for quantitative plant gene expression studies.

PubMed

Brunner, Amy M; Yakovlev, Igor A; Strauss, Steven H

2004-08-18

Real-time reverse transcription PCR (RT-PCR) has greatly improved the ease and sensitivity of quantitative gene expression studies. However, accurate measurement of gene expression with this method relies on the choice of a valid reference for data normalization. Studies rarely verify that gene expression levels for reference genes are adequately consistent among the samples used, nor compare alternative genes to assess which are most reliable for the experimental conditions analyzed. Using real-time RT-PCR to study the expression of 10 poplar (genus Populus) housekeeping genes, we demonstrate a simple method for determining the degree of stability of gene expression over a set of experimental conditions. Based on a traditional method for analyzing the stability of varieties in plant breeding, it defines measures of gene expression stability from analysis of variance (ANOVA) and linear regression. We found that the potential internal control genes differed widely in their expression stability over the different tissues, developmental stages and environmental conditions studied. Our results support that quantitative comparisons of candidate reference genes are an important part of real-time RT-PCR studies that seek to precisely evaluate variation in gene expression. The method we demonstrated facilitates statistical and graphical evaluation of gene expression stability. Selection of the best reference gene for a given set of experimental conditions should enable detection of biologically significant changes in gene expression that are too small to be revealed by less precise methods, or when highly variable reference genes are unknowingly used in real-time RT-PCR experiments.

Transcription in space--environmental vs. genetic effects on differential immune gene expression.

PubMed

Lenz, Tobias L

2015-09-01

Understanding how organisms adapt to their local environment is one of the key goals in molecular ecology. Adaptation can be achieved through qualitative changes in the coding sequence and/or quantitative changes in gene expression, where the optimal dosage of a gene's product in a given environment is being selected for. Differences in gene expression among populations inhabiting distinct environments can be suggestive of locally adapted gene regulation and have thus been studied in different species (Whitehead & Crawford ; Hodgins-Davis & Townsend ). However, in contrast to a gene's coding sequence, its expression level at a given point in time may depend on various factors, including the current environment. Although critical for understanding the extent of local adaptation, it is usually difficult to disentangle the heritable differences in gene regulation from environmental effects. In this issue of Molecular Ecology, Stutz et al. () describe an experiment in which they reciprocally transplanted three-spined sticklebacks (Gasterosteus aculeatus) between independent pairs of small and large lakes. Their experimental design allows them to attribute differences in gene expression among sticklebacks either to lake of origin or destination lake. Interestingly, they find that translocated sticklebacks show a pattern of gene expression more similar to individuals from the destination lake than to individuals from the lake of origin, suggesting that expression of the targeted genes is more strongly regulated by environmental effects than by genetics. The environmental effect by itself is not entirely surprising; however, the relative extent of it is. Especially when put in the context of local adaptation and population differentiation, as done here, these findings cast a new light onto the heritability of differential gene expression and specifically its relative importance during population divergence and ultimately ecological speciation. © 2015 John Wiley & Sons Ltd.

IGF-IEc expression is increased in secondary compared to primary foci in neuroendocrine neoplasms.

PubMed

Alexandraki, Krystallenia I; Philippou, Anastassios; Boutzios, Georgios; Theohari, Irini; Koutsilieris, Michael; Delladetsima, Ioanna Kassiani; Kaltsas, Gregory A

2017-10-03

Different Insulin-like growth factor-I (IGF-I) mRNA transcripts are produced by alternative splicing and particularly the IGF-IEc isoform has been implicated in the development and/or progression of various types of cancer. In the present study, we examined the potential role of IGF-IEc expression as a new immunohistochemical marker of aggressiveness in neuroendocrine neoplasms (NENs). We utilized immunohistochemical analysis in tissue specimens of 47 patients with NENs, to evaluate the expression of IGF-IEc (%) and Ki-67 proliferation index (%). Specimens from patients with tumors of different tissue origin, of either primary or metastatic lesions and of different grade were examined. Cytoplasmic IGF-IEc staining was found in 23 specimens of NENs or NECs: 10 pancreatic, 4 small bowel, 3 gastric, 1 lung, 1 uterine and 4 poorly differentiated of unknown primary origin. Ki-67 and IGF-IEc expression was positively correlated in all the samples studied (r=0.31, p=0.03). IGF-1Ec expression was more prevalent in specimens originating from metastatic foci with high Ki-67 compared to primary sites with low Ki-67 expression (p=0.036). These findings suggest a possible role of IGF-IEc in NEN tumorigenesis and progression to metastases that could be used as an additional new marker of a more aggressive behavior and a potential drugable target.

Molecular cloning of the Coch gene of guinea pig inner ear and its expression analysis in cultured fibrocytes of the spiral ligament.

PubMed

Li, Lishu; Ikezono, Tetsuo; Sekine, Kuwon; Shindo, Susumu; Matsumura, Tomohiro; Pawankar, Ruby; Ichimiya, Issei; Yagi, Toshiaki

2010-08-01

We have cloned guinea pig Coch cDNA and the sequence information will be useful for future molecular study combined with physiological experiments. Proper Coch gene expression appears to be dependent on the unique extracellular micro-environment of the inner ear in vivo. These results provide insight into the Coch gene expression and its regulation. To characterize the guinea pig Coch gene, we performed molecular cloning and expression analysis in the inner ear and cultured fibrocytes of the spiral ligament. The Coch cDNA was isolated using RACE. Cochlin isofoms were studied by Western blot using three different types of mammalian inner ear. The cochlear fibrocytes were cultured and characterized by immunostaining. Coch gene expression in the fibrocytes was investigated and the influence of cytokine stimulation was evaluated. The full-length 1991 bp Coch cDNA that encodes a 553 amino acid protein was isolated. The sequence had significant homology with other mammals, and the sizes of the Cochlin isoforms were identical. In the cultured fibrocytes, Coch mRNA was expressed in a very small amount and the isoform production was different, compared with the results in vivo. Cytokine stimulation did not alter the level of mRNA expression or isoform formation.

Development and characterization of a novel mouse line humanized for the intestinal peptide transporter PEPT1.

PubMed

Hu, Yongjun; Xie, Yehua; Wang, Yuqing; Chen, Xiaomei; Smith, David E

2014-10-06

The proton-coupled oligopeptide transporter PEPT1 (SLC15A1) is abundantly expressed in the small intestine, but not colon, of mammals and found to mediate the uptake of di/tripeptides and peptide-like drugs from the intestinal lumen. However, species differences have been observed in both the expression (and localization) of PEPT1 and its substrate affinity. With this in mind, the objectives of this study were to develop a humanized PEPT1 mouse model (huPEPT1) and to characterize hPEPT1 expression and functional activity in the intestines. Thus, after generating huPEPT1 mice in animals previously nulled for mouse Pept1, phenotypic, PCR, and immunoblot analyses were performed, along with in situ single-pass intestinal perfusion and in vivo oral pharmacokinetic studies with a model dipeptide, glycylsarcosine (GlySar). Overall, the huPEPT1 mice had normal survival rates, fertility, litter size, gender distribution, and body weight. There was no obvious behavioral or pathological phenotype. The mRNA and protein profiles indicated that huPEPT1 mice had substantial PEPT1 expression in all regions of the small intestine (i.e., duodenum, jejunum, and ileum) along with low but measurable expression in both proximal and distal segments of the colon. In agreement with PEPT1 expression, the in situ permeability of GlySar in huPEPT1 mice was similar to but lower than wildtype animals in small intestine, and greater than wildtype mice in colon. However, a species difference existed in the in situ transport kinetics of jejunal PEPT1, in which the maximal flux and Michaelis constant of GlySar were reduced 7-fold and 2- to 4-fold, respectively, in huPEPT1 compared to wildtype mice. Still, the in vivo function of intestinal PEPT1 appeared fully restored (compared to Pept1 knockout mice) as indicated by the nearly identical pharmacokinetics and plasma concentration-time profiles following a 5.0 nmol/g oral dose of GlySar to huPEPT1 and wildtype mice. This study reports, for the first time, the development and characterization of mice humanized for PEPT1. This novel transgenic huPEPT1 mouse model should prove useful in examining the role, relevance, and regulation of PEPT1 in diet and disease, and in the drug discovery process.

Non-targeted profiling of circulating microRNAs in women with polycystic ovary syndrome (PCOS): effects of obesity and sex hormones.

PubMed

Murri, Mora; Insenser, María; Fernández-Durán, Elena; San-Millán, José L; Luque-Ramírez, Manuel; Escobar-Morreale, Héctor F

2018-02-02

Circulating micro-ribonucleic acids (miRNAs) are small noncoding RNA molecules that influence gene transcription. We conducted the present profiling study to characterize the expression of circulating miRNAs in lean and obese patients with polycystic ovary syndrome (PCOS), the most common endocrine and metabolic disorder in premenopausal women. We selected 11 control women, 12 patients with PCOS and 12 men so that they were similar in terms of body mass index. Five control women, 6 men and 6 patients with PCOS had normal weight whereas 6 subjects per group were obese. We used miRCURY LNA™ Universal RT microRNA PCR for miRNA profiling. The expression of 38 miRNAs and was different between subjects with PCOS and male and female controls. The differences in 15 miRNAs followed a pattern suggestive of androgenization characterized by expression levels that were similar in patients with PCOS and men but were different compared with those of control women. The expression of 13 miRNAs in women with PCOS was similar to that of control women and different compared with the expression observed in men, suggesting sexual dimorphism and, lastly, we observed 5 miRNAs that were expressed differently in women with PCOS compared with both men and control women, suggesting a specific abnormality in expression associated with the syndrome. Obesity interacted with the differences in several of these miRNAs, and the expression levels of many of them correlated with the hirsutism score, sex hormones and/or indexes of obesity, adiposity and metabolic dysfunction. The present results suggest that several serum miRNAs are influenced by PCOS, sex hormones and obesity. Our findings may guide the targeted search of miRNAs as clinically relevant markers for PCOS and its association with obesity and metabolic dysfunction in future studies. Copyright © 2018. Published by Elsevier Inc.

Expression of Kv3.1b potassium channel is widespread in macaque motor cortex pyramidal cells: A histological comparison between rat and macaque.

PubMed

Soares, David; Goldrick, Isabelle; Lemon, Roger N; Kraskov, Alexander; Greensmith, Linda; Kalmar, Bernadett

2017-06-15

There are substantial differences across species in the organization and function of the motor pathways. These differences extend to basic electrophysiological properties. Thus, in rat motor cortex, pyramidal cells have long duration action potentials, while in the macaque, some pyramidal neurons exhibit short duration "thin" spikes. These differences may be related to the expression of the fast potassium channel Kv3.1b, which in rat interneurons is associated with generation of thin spikes. Rat pyramidal cells typically lack these channels, while there are reports that they are present in macaque pyramids. Here we made a systematic, quantitative comparison of the Kv3.1b expression in sections from macaque and rat motor cortex, using two different antibodies (NeuroMab, Millipore). As our standard reference, we examined, in the same sections, Kv3.1b staining in parvalbumin-positive interneurons, which show strong Kv3.1b immunoreactivity. In macaque motor cortex, a large sample of pyramidal neurons were nearly all found to express Kv3.1b in their soma membranes. These labeled neurons were identified as pyramidal based either by expression of SMI32 (a pyramidal marker), or by their shape and size, and lack of expression of parvalbumin (a marker for some classes of interneuron). Large (Betz cells), medium, and small pyramidal neurons all expressed Kv3.1b. In rat motor cortex, SMI32-postive pyramidal neurons expressing Kv3.1b were very rare and weakly stained. Thus, there is a marked species difference in the immunoreactivity of Kv3.1b in pyramidal neurons, and this may be one of the factors explaining the pronounced electrophysiological differences between rat and macaque pyramidal neurons. © 2017 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.

Identification of rat lung-specific microRNAs by micoRNA microarray: valuable discoveries for the facilitation of lung research.

PubMed

Wang, Yang; Weng, Tingting; Gou, Deming; Chen, Zhongming; Chintagari, Narendranath Reddy; Liu, Lin

2007-01-24

An important mechanism for gene regulation utilizes small non-coding RNAs called microRNAs (miRNAs). These small RNAs play important roles in tissue development, cell differentiation and proliferation, lipid and fat metabolism, stem cells, exocytosis, diseases and cancers. To date, relatively little is known about functions of miRNAs in the lung except lung cancer. In this study, we utilized a rat miRNA microarray containing 216 miRNA probes, printed in-house, to detect the expression of miRNAs in the rat lung compared to the rat heart, brain, liver, kidney and spleen. Statistical analysis using Significant Analysis of Microarray (SAM) and Tukey Honestly Significant Difference (HSD) revealed 2 miRNAs (miR-195 and miR-200c) expressed specifically in the lung and 9 miRNAs co-expressed in the lung and another organ. 12 selected miRNAs were verified by Northern blot analysis. The identified lung-specific miRNAs from this work will facilitate functional studies of miRNAs during normal physiological and pathophysiological processes of the lung.

Assessing delivery and quantifying efficacy of small interfering ribonucleic acid therapeutics in the skin using a dual-axis confocal microscope

NASA Astrophysics Data System (ADS)

Ra, Hyejun; Gonzalez-Gonzalez, Emilio; Smith, Bryan R.; Gambhir, Sanjiv S.; Kino, Gordon S.; Solgaard, Olav; Kaspar, Roger L.; Contag, Christopher H.

2010-05-01

Transgenic reporter mice and advances in imaging instrumentation are enabling real-time visualization of cellular mechanisms in living subjects and accelerating the development of novel therapies. Innovative confocal microscope designs are improving their utility for microscopic imaging of fluorescent reporters in living animals. We develop dual-axis confocal (DAC) microscopes for such in vivo studies and create mouse models where fluorescent proteins are expressed in the skin for the purpose of advancing skin therapeutics and transdermal delivery tools. Three-dimensional image volumes, through the different skin compartments of the epidermis and dermis, can be acquired in several seconds with the DAC microscope in living mice, and are comparable to histologic analyses of reporter protein expression patterns in skin sections. Intravital imaging with the DAC microscope further enables visualization of green fluorescent protein (GFP) reporter gene expression in the skin over time, and quantification of transdermal delivery of small interfering RNA (siRNA) and therapeutic efficacy. Visualization of transdermal delivery of nucleic acids will play an important role in the development of innovative strategies for treating skin pathologies.

Expression of inflammatory cytokine and inducible nitric oxide synthase genes in the small intestine and mesenteric lymph node tissues of pauci- and multibacillary sheep naturally infected with Mycobacterium avium ssp. paratuberculosis.

PubMed

Sonawane, Ganesh G; Tripathi, Bhupendra Nath

2016-12-01

Paratuberculosis (Johne's disease) is a chronic infectious granulomatous enteritis, primarily affecting ruminants, and caused by Mycobacterium avium ssp. paratuberculosis (MAP). The disease is widely prevalent throughout the world with significant economic losses. MAP has also been implicated with human Crohn's disease. There exists a strong correlation between the immune response and development of various types of pathologies in ruminants. The polarization of the immune response, which is critical to clinical outcome of the paratuberculosis infection, is controlled by the differential expression of certain cytokines and inducible nitric oxide synthase (iNOS) in Johne's disease. In previous studies, the role of different cytokines (Th1 and Th2) has been occasionally studied in sheep paratuberculosis. In the present study, we studied differential expression of interferon (IFN)-γ, interleukin (IL)-1α, IL-10, transforming growth factor (TGF)-β, iNOS, and TRAF1 genes in MAP-infected sheep and established relationship with distinct pathologies. Tissue sections (small intestine, ileocecal junction, and mesenteric lymph nodes) were collected from sheep suspected for Johne's disease and appropriately preserved for RNA extraction, polymerase chain reaction (PCR) analysis, and histopathology. Pathologic grading was done on the basis of nature and extent of cellular infiltration, granuloma formation and abundance of acid-fast bacilli. Six sheep each with pauci (PB)- and multibacillary (MB) lesions and six healthy control sheep were selected for cytokine studies. MAP in tissue extracted genomic DNA of sheep was quantified by a quantitative PCR assay. Tissue extracted RNA was reversed transcribed to prepare c-DNA from which quantitative reverse transcription PCR (qRT-PCR) was performed to amplify IFN-γ, IL-1β, IL-10, TGF-β, β-actin, TRAF1, and iNOS with Quantitect SYBR Green Master Mix. qRT-PCR data were analyzed using 2 -ΔΔCT method using β-actin gene as a control. All qRT-PCR results were compared by using one-way analysis of variance (least significance difference and Duncan tests) for p-value using SPSS (version 7.5) for expression of each gene in tissues from infected and control sheep. In the small intestine, PB sheep showed significant enhancements in the expression of IL-10, TGF-β, iNOS, and IFN-γ in comparison to similar tissues from uninfected control sheep. IL-1α expression was significantly reduced (p<0.01). The expression of IL-10 in the mesenteric lymph node (MLN) tissue of PB sheep was significantly increased (p<0.01) as compared with the control sheep. MB sheep revealed significantly enhanced expression of TGF-β mRNA and reduction in the expression of IL-1α in comparison to control sheep. In the MLN of MB sheep, the expressions of IL-10 and TGF-β were significantly (p<0.01) increased, and IFN-γ was significantly downregulated in comparison to uninfected control sheep. When the cytokine expression was compared between two distinctly infected groups, the MB sheep showed highly significant decrease (p<0.01) in the expression of iNOS and IFN-γ in the small intestine and IFN-γ in the MLN tissues. The present study indicated that IFN-γ and iNOS were found to play important role in the induction of Th1 type immune response in PB sheep. MB sheep had significant reduction in expression of IFN-γ and iNOS and elevation of IL-10 and TGF-β, which was typical of Th2 cytokine pattern. Elevated expression of IL-10 and TGF-β in PB cases possibly suggests the role of T-regulatory cells and may follow an independent mechanism not typical of Th1 pattern. In view of significantly reduced expression in both forms of the disease, IL-1α may not be an important cytokine in ovine paratuberculosis. Copyright © 2016.

snoU6 and 5S RNAs are not reliable miRNA reference genes in neuronal differentiation.

PubMed

Lim, Q E; Zhou, L; Ho, Y K; Wan, G; Too, H P

2011-12-29

Accurate profiling of microRNAs (miRNAs) is an essential step for understanding the functional significance of these small RNAs in both physiological and pathological processes. Quantitative real-time PCR (qPCR) has gained acceptance as a robust and reliable transcriptomic method to profile subtle changes in miRNA levels and requires reference genes for accurate normalization of gene expression. 5S and snoU6 RNAs are commonly used as reference genes in microRNA quantification. It is currently unknown if these small RNAs are stably expressed during neuronal differentiation. Panels of miRNAs have been suggested as alternative reference genes to 5S and snoU6 in various physiological contexts. To test the hypothesis that miRNAs may serve as stable references during neuronal differentiation, the expressions of eight miRNAs, 5S and snoU6 RNAs in five differentiating neuronal cell types were analyzed using qPCR. The stabilities of the expressions were evaluated using two complementary statistical approaches (geNorm and Normfinder). Expressions of 5S and snoU6 RNAs were stable under some but not all conditions of neuronal differentiation and thus are not suitable reference genes. In contrast, a combination of three miRNAs (miR-103, miR-106b and miR-26b) allowed accurate expression normalization across different models of neuronal differentiation. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Effects of 4-nitrophenol on expression of the ER-α and AhR signaling pathway-associated genes in the small intestine of rats.

PubMed

Tang, Juan; Song, Meiyan; Watanabe, Gen; Nagaoka, Kentaro; Rui, Xiaoli; Li, ChunMei

2016-09-01

4-Nitrophenol (PNP) is a persistent organic pollutant that was proven to be an environmental endocrine disruptor. The aim of this study was to evaluate the role of the estrogen receptor-α (ER-α) and aryl hydrocarbon receptor (AhR) signaling pathway in regulating the damage response to PNP in the small intestine of rats. Wistar-Imamichi male rats (21 d) were randomly divided into two groups: the control group and PNP group. Each group had three processes that were gavaged with PNP or vehicle daily: single dose (1 d), repeated dose (3 consecutive days) (3 d), and repeated dose with recovery (3 consecutive days and 3 recovery days) (6 d). The weight of the body, the related viscera, and small intestine were examined. Histological parameters of the small intestine and the quantity of mucus proteins secreted by small goblet cells were determined using HE staining and PAS staining. The mRNA expression of AhR, ER-α, CYP1A1, and GST was measured by real-time qPCR. In addition, we also analyzed the AhR, ER-α, and CYP1A1 expression in the small intestine by immunohistochemical staining. The small intestines histologically changed in the PNP-treated rat and the expression of AhR, CYP1A1, and GST was increased. While ER-α was significantly decreased in the small intestine, simultaneously, when rats were exposed to a longer PNP treatment, the damages disappeared. Our results demonstrate that PNP has an effect on the expression of AhR signaling pathway genes, AhR, CYP1A1, and GST, and ER-α in the rat small intestine. Copyright © 2016 Elsevier Ltd. All rights reserved.

High-Throughput Genetic Analysis and Combinatorial Chiral Separations Based on Capillary Electrophoresis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhong, Wenwan

2003-01-01

Capillary electrophoresis (CE) offers many advantages over conventional analytical methods, such as speed, simplicity, high resolution, low cost, and small sample consumption, especially for the separation of enantiomers. However, chiral method developments still can be time consuming and tedious. They designed a comprehensive enantioseparation protocol employing neutral and sulfated cyclodextrins as chiral selectors for common basic, neutral, and acidic compounds with a 96-capillary array system. By using only four judiciously chosen separation buffers, successful enantioseparations were achieved for 49 out of 54 test compounds spanning a large variety of pKs and structures. Therefore, unknown compounds can be screened in thismore » manner to identify optimal enantioselective conditions in just one rn. In addition to superior separation efficiency for small molecules, CE is also the most powerful technique for DNA separations. Using the same multiplexed capillary system with UV absorption detection, the sequence of a short DNA template can be acquired without any dye-labels. Two internal standards were utilized to adjust the migration time variations among capillaries, so that the four electropherograms for the A, T, C, G Sanger reactions can be aligned and base calling can be completed with a high level of confidence. the CE separation of DNA can be applied to study differential gene expression as well. Combined with pattern recognition techniques, small variations among electropherograms obtained by the separation of cDNA fragments produced from the total RNA samples of different human tissues can be revealed. These variations reflect the differences in total RNA expression among tissues. Thus, this Ce-based approach can serve as an alternative to the DNA array techniques in gene expression analysis.« less

Aberrant expression of miR-141 and nuclear receptor small heterodimer partner in clinical samples of prostate cancer.

PubMed

Khorasani, Maryam; Teimoori-Toolabi, Ladan; Farivar, Taghi Naserpour; Asgari, Mojgan; Abolhasani, Maryam; Shahrokh, Hossein; Afgar, Ali; Kalantari, Elham; Peymani, Amir; Mahdian, Reza

2018-01-01

Prostate cancer (PCa) is the second most common cancer in men worldwide. Currently, prostate-specific antigen (PSA) test and digital rectal exam are the main screening tests used for PCa diagnosis. However, due to the low specificity of these tests, new alternative biomarkers such as deregulated RNAs and microRNAs have been implemented. Aberrant expressions of small heterodimer partner gene (SHP, NR0B2) and mir-141 are reported in various cancers. The aim of this study was to investigate the SHP and miR-141 expression level in tissue samples of prostate cancer. The expression level of SHP gene and miR-141 was assessed by real time PCR and their relative amounts were calculated by the Δ⁢ΔCT method. Also, IHC technique was used to determine the expression level of SHP protein. The miR-141 was significantly up-regulated in the samples of metastatic tumors compared to localized tumor samples (P< 0.001, 31.17-fold change). Tumor samples showed lower SHP mRNA expression levels than BPH samples (p= 0.014, 4.7-fold change). The results of paired t-test analysis showed there was no significant difference between the SHP gene expression in PCa samples and their matched tumor-adjacent normal tissue (p= 0.5). The data obtained in our study confirm the involvement of miR-141 in PCa progression and metastasis. These effects could be mediated by AR via down-regulation of its co-repressor protein, i.e., SHP.

High-throughput sequencing of small RNAs from pollen and silk and characterization of miRNAs as candidate factors involved in pollen-silk interactions in maize.

PubMed

Li, Xiao Ming; Sang, Ya Lin; Zhao, Xiang Yu; Zhang, Xian Sheng

2013-01-01

In angiosperms, successful pollen-pistil interactions are the prerequisite and guarantee of subsequent fertilization and seed production. Recent profile analyses have helped elucidate molecular mechanisms underlying these processes at both transcriptomic and proteomic levels, but the involvement of miRNAs in pollen-pistil interactions is still speculative. In this study, we sequenced four small RNA libraries derived from mature pollen, in vitro germinated pollen, mature silks, and pollinated silks of maize (Zea mays L.). We identified 161 known miRNAs belonging to 27 families and 82 novel miRNAs. Of these, 40 conserved and 16 novel miRNAs showed different expression levels between mature and germinated pollen, and 30 conserved and eight novel miRNAs were differentially expressed between mature and pollinated silks. As candidates for factors associated with pollen-silk (pistil) interactions, expression patterns of the two sets of differentially expressed miRNAs were confirmed by stem-loop real-time RT-PCR. Transcript levels of 22 predicted target genes were also validated using real-time RT-PCR; most of these exhibited expression patterns contrasting with those of their corresponding miRNAs. In addition, GO analysis of target genes of differentially expressed miRNAs revealed that functional categories related to auxin signal transduction and gene expression regulation were overrepresented. These results suggest that miRNA-mediated auxin signal transduction and transcriptional regulation have roles in pollen-silk interactions. The results of our study provide novel information for understanding miRNA regulatory roles in pollen-pistil interactions.

The human TRPV6 channel protein is associated with cyclophilin B in human placenta.

PubMed

Stumpf, Tobias; Zhang, Qi; Hirnet, Daniela; Lewandrowski, Urs; Sickmann, Albert; Wissenbach, Ulrich; Dörr, Janka; Lohr, Christian; Deitmer, Joachim W; Fecher-Trost, Claudia

2008-06-27

Transcellular calcium transport in the kidney, pancreas, small intestine, and placenta is partly mediated by transient receptor potential (TRP) channels. The highly selective TRPV6 calcium channel protein is most likely important for the calcium transfer in different specialized epithelial cells. In the human placenta the protein is expressed in trophoblast tissue, where it is implicated in the transepithelial calcium transfer from mother to the fetus. We enriched the TRPV6 channel protein endogenously expressed in placenta together with annexin A2 and cyclophilin B (CypB), which is a member of the huge immunophilin family. In the human placenta TRPV6 and CypB are mainly located intracellularly in the syncytiotrophoblast layer, but a small amount of the mature glycosylated TRPV6 channel protein and CypB is also expressed in microvilli apical membranes, the fetomaternal barrier. To understand the role of CypB on the TRPV6 channel function, we evaluated the effect of CypB co-expression on TRPV6-mediated calcium uptake into Xenopus laevis oocytes expressing TRPV6. A significant increase of TRPV6-mediated calcium uptake was observed after CypB/TRPV6 co-expression. This stimulatory effect of CypB was reversed by the immunosuppressive drug cyclosporin A, which inhibits the enzymatic activity of CypB. Cyclosporin A had no significant effect on TRPV6 and CypB protein expression levels in the oocytes. In summary, our results establish CypB as a new TRPV6 accessory protein with potential involvement in TRPV6 channel activation through its peptidyl-prolyl cis/trans isomerase activity.

Prognostic impact of alternative splicing-derived hMENA isoforms in resected, node-negative, non-small-cell lung cancer

PubMed Central

Sperduti, Isabella; Iapicca, Pierluigi; Visca, Paolo; Alessandrini, Gabriele; Antoniani, Barbara; Pilotto, Sara; Ludovini, Vienna; Vannucci, Jacopo; Bellezza, Guido; Sidoni, Angelo; Tortora, Giampaolo; Radisky, Derek C.; Crinò, Lucio; Cognetti, Francesco; Facciolo, Francesco; Mottolese, Marcella

2014-01-01

Risk assessment and treatment choice remain a challenge in early non-small-cell lung cancer (NSCLC). Alternative splicing is an emerging source for diagnostic, prognostic and therapeutic tools. Here, we investigated the prognostic value of the actin cytoskeleton regulator hMENA and its isoforms, hMENA11a and hMENAΔv6, in early NSCLC. The epithelial hMENA11a isoform was expressed in NSCLC lines expressing E-CADHERIN and was alternatively expressed with hMENAΔv6. Enforced expression of hMENAΔv6 or hMENA11a increased or decreased the invasive ability of A549 cells, respectively. hMENA isoform expression was evaluated in 248 node-negative NSCLC. High pan-hMENA and low hMENA11a were the only independent predictors of shorter disease-free and cancer-specific survival, and low hMENA11a was an independent predictor of shorter overall survival, at multivariate analysis. Patients with low pan-hMENA/high hMENA11a expression fared significantly better (P≤0.0015) than any other subgroup. Such hybrid variable was incorporated with T-size and number of resected lymph nodes into a 3-class-risk stratification model, which strikingly discriminated between different risks of relapse, cancer-related death, and death. The model was externally validated in an independent dataset of 133 patients. Relative expression of hMENA splice isoforms is a powerful prognostic factor in early NSCLC, complementing clinical parameters to accurately predict individual patient risk. PMID:25373410

Identification of Mouse Serum miRNA Endogenous References by Global Gene Expression Profiles

PubMed Central

Mi, Qing-Sheng; Weiland, Matthew; Qi, Rui-Qun; Gao, Xing-Hua; Poisson, Laila M.; Zhou, Li

2012-01-01

MicroRNAs (miRNAs) are recently discovered small non-coding RNAs and can serve as serum biomarkers for disease diagnosis and prognoses. Lack of reliable serum miRNA endogenous references for normalization in miRNA gene expression makes single miRNA assays inaccurate. Using TaqMan® real-time PCR miRNA arrays with a global gene expression normalization strategy, we have analyzed serum miRNA expression profiles of 20 female mice of NOD/ShiLtJ (n = 8), NOR/LtJ (n = 6), and C57BL/6J (n = 6) at different ages and disease conditions. We identified five miRNAs, miR-146a, miR-16, miR-195, miR-30e and miR-744, to be stably expressed in all strains, which could serve as mouse serum miRNA endogenous references for single assay experiments. PMID:22348064

Long non‑coding RNA AK001796 contributes to cisplatin resistance of non‑small cell lung cancer.

PubMed

Liu, Bin; Pan, Chun-Feng; Ma, Teng; Wang, Jun; Yao, Guo-Liang; Wei, Ke; Chen, Yi-Jiang

2017-10-01

Cisplatin (DDP)‑based chemotherapy is the most widely used therapy for non‑small cell lung cancer (NSCLC). However, the existence of chemoresistance has become a major limitation in its efficacy. Long non‑coding RNAs (lncRNAs) have been shown to be involved in chemotherapy drug resistance. The aim of the present study was to investigate the biological role of lncRNA AK001796 in cisplatin‑resistant NSCLC A549/DDP cells. Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) analysis was performed to monitor the differences in the expression of AK001796 in cisplatin-resistant (A549/DDP) cells and parental A549 cells. Cellular sensitivity to cisplatin and cell viability were examined using an MTT assay. Cell apoptosis and cell cycle distribution were measured using flow cytometry. The expression levels of cell cycle proteins cyclin C (CCNC), baculoviral IAP repeat containing 5 (BIRC5), cyclin‑dependent kinase 1 (CDK1) and G2 and S phase‑expressed 1 (GTSE1) were assessed using RT‑qPCR and western blot analyses. It was found that the expression of AK001796 was increased in A549/DDP cells, compared with that in A549 cells. The knockdown of AK001796 by small interfering RNA reduced cellular cisplatin resistance and cell viability, and resulted in cell‑cycle arrest, with a marked increase in the proportion of A549/DDP cells in the G0/G1 phase. By contrast, the knockdown of AK001796 increased the number of apoptotic cancer cells during cisplatin treatment. It was also shown that the knockdown of AK001796 positively induced the expression of cell apoptosis‑associated factors, CCNC and BIRC5, and suppressed the expression of cell cycle‑associated factors, CDK1 and GTSE5. Taken together, these findings indicated that lncRNA AK001796 increased the resistance of NSCLC cells to cisplatin through regulating cell apoptosis and cell proliferation, and thus provides an attractive therapeutic target for NSCLC.

Identification and molecular characterization of a trans-acting small interfering RNA producing locus regulating leaf rust responsive gene expression in wheat (Triticum aestivum L.).

PubMed

Dutta, Summi; Kumar, Dhananjay; Jha, Shailendra; Prabhu, Kumble Vinod; Kumar, Manish; Mukhopadhyay, Kunal

2017-11-01

A novel leaf rust responsive ta-siRNA-producing locus was identified in wheat showing similarity to 28S rRNA and generated four differentially expressing ta-siRNAs by phasing which targeted stress responsive genes. Trans-acting-small interfering RNAs (Ta-siRNAs) are plant specific molecules generally involved in development and are also stress responsive. Ta-siRNAs identified in wheat till date are all responsive to abiotic stress only. Wheat cultivation is severely affected by rusts and leaf rust particularly affects grain filling. This study reports a novel ta-siRNA producing locus (TAS) in wheat which is a segment of 28S ribosomal RNA but shows differential expression during leaf rust infestation. Four small RNA libraries prepared from wheat Near Isogenic Lines were treated with leaf rust pathogen and compared with untreated controls. A TAS with the ability to generate four ta-siRNAs by phasing events was identified along with the microRNA TamiR16 as the phase initiator. The targets of the ta-siRNAs included α-gliadin, leucine rich repeat, trans-membrane proteins, glutathione-S-transferase, and fatty acid desaturase among others, which are either stress responsive genes or are essential for normal growth and development of plants. Expression of the TAS, its generated ta-siRNAs, and their target genes were profiled at five different time points after pathogen inoculation of susceptible and resistant wheat isolines and compared with mock-inoculated controls. Comparative analysis of expression unveiled differential and reciprocal relationship as well as discrete patterns between susceptible and resistant isolines. The expression profiles of the target genes of the identified ta-siRNAs advocate more towards effector triggered susceptibility favouring pathogenesis. The study helps in discerning the functions of wheat genes regulated by ta-siRNAs in response to leaf rust.

Cadmium modulates adipocyte functions in metallothionein-null mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kawakami, Takashige; Nishiyama, Kaori; Kadota, Yoshito

2013-11-01

Our previous study has demonstrated that exposure to cadmium (Cd), a toxic heavy metal, causes a reduction of adipocyte size and the modulation of adipokine expression. To further investigate the significance of the Cd action, we studied the effect of Cd on the white adipose tissue (WAT) of metallothionein null (MT{sup −/−}) mice, which cannot form atoxic Cd–MT complexes and are used for evaluating Cd as free ions, and wild type (MT{sup +/+}) mice. Cd administration more significantly reduced the adipocyte size of MT{sup −/−} mice than that of MT{sup +/+} mice. Cd exposure also induced macrophage recruitment to WATmore » with an increase in the expression level of Ccl2 (MCP-1) in the MT{sup −/−} mice. The in vitro exposure of Cd to adipocytes induce triglyceride release into culture medium, decrease in the expression levels of genes involved in fatty acid synthesis and lipid hydrolysis at 24 h, and at 48 h increase in phosphorylation of the lipid-droplet-associated protein perilipin, which facilitates the degradation of stored lipids in adipocytes. Therefore, the reduction in adipocyte size by Cd may arise from an imbalance between lipid synthesis and lipolysis. In addition, the expression levels of leptin, adiponectin and resistin decreased in adipocytes. Taken together, exposure to Cd may induce unusually small adipocytes and modulate the expression of adipokines differently from the case of physiologically small adipocytes, and may accelerate the risk of developing insulin resistance and type 2 diabetes. - Highlights: • Cd causes a marked reduction in adipocyte size in MT-null mice. • Cd enhances macrophage migration into adipose tissue and disrupt adipokine secretion. • MT gene alleviates Cd-induced adipocyte dysfunctions. • Cd enhances the degradation of stored lipids in adipocytes, mediated by perilipin. • Cd induces unusually small adipocytes and the abnormal expression of adipokines.« less

Regulation of gene expression in intervertebral disc cells by low and high hydrostatic pressure.

PubMed

Neidlinger-Wilke, Cornelia; Würtz, Karin; Urban, Jill P G; Börm, Wolfgang; Arand, Markus; Ignatius, Anita; Wilke, Hans-Joachim; Claes, Lutz E

2006-08-01

Intervertebral disc structures are exposed to wide ranges of intradiscal hydrostatic pressure during different loading exercises and are at their minimum during lying or relaxed sitting and at maximum during lifting weights with a round back. We hypothesize that these different loading magnitudes influence the intervertebral disc (IVD) by alteration of disc matrix turnover depending on their magnitudes. Therefore the aim of this study was to assess changes in gene expression of human nucleus cells after the application of low hydrostatic pressure (0.25 MPa) and high hydrostatic pressure (2.5 MPa). IVD cells isolated from the nucleus of human (n = 18) and bovine (n = 24 from four animals) disc biopsies were seeded into three-dimensional collagen type-I matrices and exposed to the different loading magnitudes by specially developed pressure chambers. The lower pressure range (0.25 MPa, 30 min, 0.1 Hz) was applied with a recently published device by using an external compression cylinder. For the application of higher loads (2.5 MPa, 30 min, 0.1 Hz) the cell-loaded collagen gels were sealed into sterile bags with culture medium and stimulated in a newly developed water-filled compression cylinder by using a loading frame. These methods allowed the comparison of loading regimes in a wide physiological range under an equal three-dimensional culture conditions. Cells were harvested 24 h after the end of stimulation and changes in the expression of genes known to influence IVD matrix turnover (collagen-I, collagen-II, aggrecan, MMP1, MMP2, MMP3, MMP13) were analyzed by real-time RT-PCR. A Wilcoxon signed-rank test(1) and a Wilcoxon 2-sample test(2) were performed to detect differences between the stimulated and control samples(1) and differences between low and high hydrostatic pressure(2). Multiple testing was considered by adjusting the p value appropriately. Both regimes of hydrostatic pressure influenced gene expression in nucleus cells with opposite tendencies for the matrix forming proteins aggrecan and collagen type-I in response to the two different pressure magnitudes: Low hydrostatic-pressure (0.25 MPa) tended to increase collagen-I and aggrecan expression of human nucleus cells (P < 0.05) but only to a small degree. High hydrostatic pressure (2.5 MPa) tended to decrease gene expression of all anabolic proteins with significant effects on aggrecan expression of nucleus cells (P = 0.004). Low hydrostatic pressure had no influence on the expression of matrix metalloproteinases (MMP1, MMP2, MMP3 and MMP13). In contrast, high hydrostatic pressure tended to increase the expression of MMP1, MMP3 and MMP13 of human nucleus cells with high individual-individual variations. The decreased expression of aggrecan (P = 0.008) and collagen type II (P = 0.023) and the increased MMP3 expression (P = 0.008) in response to high hydrostatic pressure could be confirmed in additional experiments with bovine nucleus cells. These results suggest that hydrostatic pressure as one of the physiological stimuli of the IVD may influence matrix turnover in a magnitude dependent way. Low hydrostatic pressure (0.25 MPa) has quite small influences with a tendency to anabolic effects, whereas high hydrostatic pressure (2.5 MPa) tends to decrease the matrix protein expression with a tendency to increase some matrix-turnover enzymes. Therefore, hydrostatic pressure may regulate disc matrix turnover in a dose-dependent way.

Identification of microRNAs in the Toxigenic Dinoflagellate Alexandrium catenella by High-Throughput Illumina Sequencing and Bioinformatic Analysis

PubMed Central

Geng, Huili; Sui, Zhenghong; Zhang, Shu; Du, Qingwei; Ren, Yuanyuan; Liu, Yuan; Kong, Fanna; Zhong, Jie; Ma, Qingxia

2015-01-01

Micro-ribonucleic acids (miRNAs) are a large group of endogenous, tiny, non-coding RNAs consisting of 19–25 nucleotides that regulate gene expression at either the transcriptional or post-transcriptional level by mediating gene silencing in eukaryotes. They are considered to be important regulators that affect growth, development, and response to various stresses in plants. Alexandrium catenella is an important marine toxic phytoplankton species that can cause harmful algal blooms (HABs). To date, identification and function analysis of miRNAs in A. catenella remain largely unexamined. In this study, high-throughput sequencing was performed on A. catenella to identify and quantitatively profile the repertoire of small RNAs from two different growth phases. A total of 38,092,056 and 32,969,156 raw reads were obtained from the two small RNA libraries, respectively. In total, 88 mature miRNAs belonging to 32 miRNA families were identified. Significant differences were found in the member number, expression level of various families, and expression abundance of each member within a family. A total of 15 potentially novel miRNAs were identified. Comparative profiling showed that 12 known miRNAs exhibited differential expression between the lag phase and the logarithmic phase. Real-time quantitative RT-PCR (qPCR) was performed to confirm the expression of two differentially expressed miRNAs that were one up-regulated novel miRNA (aca-miR-3p-456915), and one down-regulated conserved miRNA (tae-miR159a). The expression trend of the qPCR assay was generally consistent with the deep sequencing result. Target predictions of the 12 differentially expressed miRNAs resulted in 1813target genes. Gene ontology (GO) analysis and the Kyoto Encyclopedia of Genes and Genomes pathway database (KEGG) annotations revealed that some miRNAs were associated with growth and developmental processes of the alga. These results provide insights into the roles that miRNAs play in the growth of A. catenella, and they provide the basis for further studies of the molecular mechanisms that underlie bloom growth in red tides species. PMID:26398216

Performances of survival, feeding behavior, and gene expression in aphids reveal their different fitness to host alteration

PubMed Central

Lu, Hong; Yang, Pengcheng; Xu, Yongyu; Luo, Lan; Zhu, Junjie; Cui, Na; Kang, Le; Cui, Feng

2016-01-01

Insect populations feeding on different plant species are under selection pressure to adapt to these differences. A study integrating elements of the ecology, behavior, and gene expression of aphids on different host plants has not yet been well-explored. The present study explores the relationship between host fitness and survival, feeding behavior, and salivary gland gene expression of a pea (Pisum sativum) host race of Acyrthosiphon pisum feeding on a common host Vicia faba and on three genetically-related hosts (Vicia villosa, Medicago truncatula, and Medicago sativa). Life table data indicated that aphids on non-favored hosts exhibited small size, low reproduction rate, slow population increase and individual development, and long lifespan. Electrical penetration graph results showed that the aphids spent significantly less time in passive ingestion of phloem sap on all non-preferred host plants before acclimation. After a period of acclimation on M. truncatula and V. villosa, pea host race individuals showed improved feeding behavior. No individuals of the pea host race completed its life history on M. sativa. Interestingly, the number of host-specific differentially-expressed salivary gland genes was negatively correlated with the fitness of aphids on this host plant. This study provided important cues in host plant specialization in aphids. PMID:26758247

A preliminary result of three-dimensional microarray technology to gene analysis with endoscopic ultrasound-guided fine-needle aspiration specimens and pancreatic juices

PubMed Central

2010-01-01

Background Analysis of gene expression and gene mutation may add information to be different from ordinary pathological tissue diagnosis. Since samples obtained endoscopically are very small, it is desired that more sensitive technology is developed for gene analysis. We investigated whether gene expression and gene mutation analysis by newly developed ultra-sensitive three-dimensional (3D) microarray is possible using small amount samples from endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) specimens and pancreatic juices. Methods Small amount samples from 17 EUS-FNA specimens and 16 pancreatic juices were obtained. After nucleic acid extraction, the samples were amplified with labeling and analyzed by the 3D microarray. Results The analyzable rate with the microarray was 46% (6/13) in EUS-FNA specimens of RNAlater® storage, and RNA degradations were observed in all the samples of frozen storage. In pancreatic juices, the analyzable rate was 67% (4/6) in frozen storage samples and 20% (2/10) in RNAlater® storage. EUS-FNA specimens were classified into cancer and non-cancer by gene expression analysis and K-ras codon 12 mutations were also detected using the 3D microarray. Conclusions Gene analysis from small amount samples obtained endoscopically was possible by newly developed 3D microarray technology. High quality RNA from EUS-FNA samples were obtained and remained in good condition only using RNA stabilizer. In contrast, high quality RNA from pancreatic juice samples were obtained only in frozen storage without RNA stabilizer. PMID:20416107

[Small airway diseases and immune deficiency].

PubMed

Burgel, P-R; Bergeron, A; Knoop, C; Dusser, D

2016-02-01

Innate or acquired immune deficiency may show respiratory manifestations, often characterized by small airway involvement. The purpose of this article is to provide an overview of small airway disease across the major causes of immune deficiency. In patients with common variable immune deficiency, recurrent lower airway infections may lead to bronchiolitis and bronchiectasis. Follicular and/or granulomatous bronchiolitis of unknown origin may also occur. Bronchiolitis obliterans is the leading cause of death after the first year in patients with lung transplantation. Bronchiolitis obliterans also occurs in patients with allogeneic haematopoietic stem cell transplantation, especially in the context of systemic graft-versus-host disease. Small airway diseases have different clinical expression and pathophysiology across various causes of immune deficiency. A better understanding of small airways disease pathogenesis in these settings may lead to the development of novel targeted therapies. Copyright © 2015 SPLF. Published by Elsevier Masson SAS. All rights reserved.

Cell culture-based profiling across mammals reveals DNA repair and metabolism as determinants of species longevity.

PubMed

Ma, Siming; Upneja, Akhil; Galecki, Andrzej; Tsai, Yi-Miau; Burant, Charles F; Raskind, Sasha; Zhang, Quanwei; Zhang, Zhengdong D; Seluanov, Andrei; Gorbunova, Vera; Clish, Clary B; Miller, Richard A; Gladyshev, Vadim N

2016-11-22

Mammalian lifespan differs by >100 fold, but the mechanisms associated with such longevity differences are not understood. Here, we conducted a study on primary skin fibroblasts isolated from 16 species of mammals and maintained under identical cell culture conditions. We developed a pipeline for obtaining species-specific ortholog sequences, profiled gene expression by RNA-seq and small molecules by metabolite profiling, and identified genes and metabolites correlating with species longevity. Cells from longer lived species up-regulated genes involved in DNA repair and glucose metabolism, down-regulated proteolysis and protein transport, and showed high levels of amino acids but low levels of lysophosphatidylcholine and lysophosphatidylethanolamine. The amino acid patterns were recapitulated by further analyses of primate and bird fibroblasts. The study suggests that fibroblast profiling captures differences in longevity across mammals at the level of global gene expression and metabolite levels and reveals pathways that define these differences.

Use of lambdagt11 to isolate genes for two pseudorabies virus glycoproteins with homology to herpes simplex virus and varicella-zoster virus glycoproteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Petrovskis, E.A.; Timmins, J.G.; Post, L.E.

1986-10-01

A library of pseudorabies virus (PRV) DNA fragments was constructed in the expression cloning vector lambdagt11. The library was screened with antisera which reacted with mixtures of PRV proteins to isolate recombinant bacteriophages expressing PRV proteins. By the nature of the lambdagt11 vector, the cloned proteins were expressed in Escherichia coli as ..beta..-galactosidase fusion proteins. The fusion proteins from 35 of these phages were purified and injected into mice to raise antisera. The antisera were screened by several different assays, including immunoprecipitation of (/sup 14/C)glucosamine-labeled PRV proteins. This method identified phages expressing three different PRV glycoproteins: the secreted glycoprotein, gX;more » gI; and a glycoprotein that had not been previously identified, which we designate gp63. The gp63 and gI genes map adjacent to each other in the small unique region of the PRV genome. The DNA sequence was determined for the region of the genome encoding gp63 and gI. It was found that gp63 has a region of homology with a herpes simplex virus type 1 (HSV-1) protein, encoded by US7, and also with varicella-zoster virus (VZV) gpIV. The gI protein sequence has a region of homology with HSV-1 gE and VZV gpI. It is concluded that PRV, HSV, and VZV all have a cluster of homologous glycoprotein genes in the small unique components of their genomes and that the organization of these genes is conserved.« less

Embedding siRNA sequences targeting Apolipoprotein B100 in shRNA and miRNA scaffolds results in differential processing and in vivo efficacy

PubMed Central

Maczuga, Piotr; Lubelski, Jacek; van Logtenstein, Richard; Borel, Florie; Blits, Bas; Fakkert, Erwin; Costessi, Adalberto; Butler, Derek; van Deventer, Sander; Petry, Harald; Koornneef, Annemart; Konstantinova, Pavlina

2013-01-01

Overexpression of short hairpin RNA (shRNA) often causes cytotoxicity and using microRNA (miRNA) scaffolds can circumvent this problem. In this study, identically predicted small interfering RNA (siRNA) sequences targeting apolipoprotein B100 (siApoB) were embedded in shRNA (shApoB) or miRNA (miApoB) scaffolds and a direct comparison of the processing and long-term in vivo efficacy was performed. Next generation sequencing of small RNAs originating from shApoB- or miApoB-transfected cells revealed substantial differences in processing, resulting in different siApoB length, 5′ and 3′ cleavage sites and abundance of the guide or passenger strands. Murine liver transduction with adeno-associated virus (AAV) vectors expressing shApoB or miApoB resulted in high levels of siApoB expression associated with strong decrease of plasma ApoB protein and cholesterol. Expression of miApoB from the liver-specific LP1 promoter was restricted to the liver, while the H1 promoter-expressed shApoB was ectopically present. Delivery of 1 × 1011 genome copies AAV-shApoB or AAV-miApoB led to a gradual loss of ApoB and plasma cholesterol inhibition, which was circumvented by delivering a 20-fold lower vector dose. In conclusion, incorporating identical siRNA sequences in shRNA or miRNA scaffolds results in differential processing patterns and in vivo efficacy that may have serious consequences for future RNAi-based therapeutics. PMID:23089734

The Impact of Repeated Freeze-Thaw Cycles on the Quality of Biomolecules in Four Different Tissues.

PubMed

Ji, Xiaoli; Wang, Min; Li, Lingling; Chen, Fang; Zhang, Yanyang; Li, Qian; Zhou, Junmei

2017-10-01

High-quality biosamples are valuable resources for biomedical research. However, some tissues are stored without being sectioned into small aliquots and have to undergo repeated freeze-thaw cycles throughout prolonged experimentation. Little is known regarding the effects of repeated freeze-thaw cycles on the quality of biomolecules in tissues. The aim of this study was to evaluate the impact of repeated freeze-thaw (at room temperature or on ice) cycles on biomolecules and gene expression in four different types of tissues. Each fresh tissue was sectioned into seven aliquots and snap-frozen before undergoing repeated freeze-thaw cycles at room temperature or on ice. Biomolecules were extracted and analyzed. Both relative and absolute quantification were used to detect the changes in gene expression. The results indicated that the impact of repeated freeze-thaw cycles on RNA integrity varied by tissue type. Gene expression, including the housekeeping gene, was affected in RNA-degraded samples according to absolute quantification rather than relative quantification. Furthermore, our results suggest that thawing on ice could protect RNA integrity compared with thawing at room temperature. No obvious degradation of protein or DNA was observed with repeated freeze-thaw cycles either at room temperature or on ice. This research provides ample evidence for the necessity of sectioning fresh tissues into small aliquots before snap-freezing, thus avoiding degradation of RNA and alteration of gene expression resulting from repeated freeze-thaw cycles. For frozen tissue samples that were already in storage and had to be used repeatedly during their lifecycle, thawing on ice or sectioned at ultralow temperature is recommended.

Growth of embryo and gene expression of nutrient transporters in the small intestine of the domestic pigeon (Columba livia)*

PubMed Central

Chen, Ming-xia; Li, Xiang-guang; Yang, Jun-xian; Gao, Chun-qi; Wang, Bin; Wang, Xiu-qi; Yan, Hui-chao

2015-01-01

The objective of this study was to investigate the relationship between gene expression of nutrient (amino acid, peptide, sodium and proton) transporters in the small intestine and embryonic growth in domestic pigeons (Columba livia). One hundred and twenty-five fertilized eggs were randomly assigned into five groups and were incubated under optimal conditions (temperature of 38.1 °C and relative humidity of 55%). Twenty embryos/birds from each group were sacrificed by cervical dislocation on embryonic day (E) 9, 11, 13, 15 and day of hatch (DOH). The eggs, embryos (without yolk sac), and organs (head, brain, heart, liver, lungs, kidney, gizzard, small intestine, legs, and thorax) were dissected, cleaned, and weighed. Small intestine samples were collected for RNA isolation. The mRNA abundance of intestinal nutrient transporters was evaluated by real-time reverse transcription-polymerase chain reaction (RT-PCR). We classified these ten organs into four types according to the changes in relative weight during embryonic development. In addition, the gene expression of nutrient transporters was differentially regulated by embryonic day. The mRNA abundances of b0,+AT, EAAT3, y+LAT2, PepT1, LAT4, NHE2, and NHE3 increased linearly with age, whereas mRNA abundances of CAT1, CAT2, LAT1, EAAT2, SNAT1, and SNAT2 were increased to higher levels on E9 or E11 and then decreased to lower levels until DOH. The results of correlation analysis showed that the gene expressions of b0,+AT, EAAT3, PepT1, LAT4, NHE2, NHE3, and y+LAT2 had positive correlations with body weight (0.71

An Integrated Bioinformatics Approach Identifies Elevated Cyclin E2 Expression and E2F Activity as Distinct Features of Tamoxifen Resistant Breast Tumors

PubMed Central

Huang, Lei; Zhao, Shuangping; Frasor, Jonna M.; Dai, Yang

2011-01-01

Approximately half of estrogen receptor (ER) positive breast tumors will fail to respond to endocrine therapy. Here we used an integrative bioinformatics approach to analyze three gene expression profiling data sets from breast tumors in an attempt to uncover underlying mechanisms contributing to the development of resistance and potential therapeutic strategies to counteract these mechanisms. Genes that are differentially expressed in tamoxifen resistant vs. sensitive breast tumors were identified from three different publically available microarray datasets. These differentially expressed (DE) genes were analyzed using gene function and gene set enrichment and examined in intrinsic subtypes of breast tumors. The Connectivity Map analysis was utilized to link gene expression profiles of tamoxifen resistant tumors to small molecules and validation studies were carried out in a tamoxifen resistant cell line. Despite little overlap in genes that are differentially expressed in tamoxifen resistant vs. sensitive tumors, a high degree of functional similarity was observed among the three datasets. Tamoxifen resistant tumors displayed enriched expression of genes related to cell cycle and proliferation, as well as elevated activity of E2F transcription factors, and were highly correlated with a Luminal intrinsic subtype. A number of small molecules, including phenothiazines, were found that induced a gene signature in breast cancer cell lines opposite to that found in tamoxifen resistant vs. sensitive tumors and the ability of phenothiazines to down-regulate cyclin E2 and inhibit proliferation of tamoxifen resistant breast cancer cells was validated. Our findings demonstrate that an integrated bioinformatics approach to analyze gene expression profiles from multiple breast tumor datasets can identify important biological pathways and potentially novel therapeutic options for tamoxifen-resistant breast cancers. PMID:21789246

Normalization of gene expression measurement of tissue samples obtained by transurethral resection of bladder tumors.

PubMed

Pop, Laura A; Pileczki, Valentina; Cojocneanu-Petric, Roxana M; Petrut, Bogdan; Braicu, Cornelia; Jurj, Ancuta M; Buiga, Rares; Achimas-Cadariu, Patriciu; Berindan-Neagoe, Ioana

2016-01-01

Sample processing is a crucial step for all types of genomic studies. A major challenge for researchers is to understand and predict how RNA quality affects the identification of transcriptional differences (by introducing either false-positive or false-negative errors). Nanotechnologies help improve the quality and quantity control for gene expression studies. The study was performed on 14 tumor and matched normal pairs of tissue from patients with bladder urothelial carcinomas. We assessed the RNA quantity by using the NanoDrop spectrophotometer and the quality by nano-microfluidic capillary electrophoresis technology provided by Agilent 2100 Bioanalyzer. We evaluated the amplification status of three housekeeping genes and one small nuclear RNA gene using the ViiA 7 platform, with specific primers. Every step of the sample handling protocol, which begins with sample harvest and ends with the data analysis, is of utmost importance due to the fact that it is time consuming, labor intensive, and highly expensive. High temperature of the surgical procedure does not affect the small nucleic acid sequences in comparison with the mRNA. Gene expression is clearly affected by the RNA quality, but less affected in the case of small nuclear RNAs. We proved that the high-temperature, highly invasive transurethral resection of bladder tumor procedure damages the tissue and affects the integrity of the RNA from biological specimens.

Normalization of gene expression measurement of tissue samples obtained by transurethral resection of bladder tumors

PubMed Central

Pop, Laura A; Pileczki, Valentina; Cojocneanu-Petric, Roxana M; Petrut, Bogdan; Braicu, Cornelia; Jurj, Ancuta M; Buiga, Rares; Achimas-Cadariu, Patriciu; Berindan-Neagoe, Ioana

2016-01-01

Background Sample processing is a crucial step for all types of genomic studies. A major challenge for researchers is to understand and predict how RNA quality affects the identification of transcriptional differences (by introducing either false-positive or false-negative errors). Nanotechnologies help improve the quality and quantity control for gene expression studies. Patients and methods The study was performed on 14 tumor and matched normal pairs of tissue from patients with bladder urothelial carcinomas. We assessed the RNA quantity by using the NanoDrop spectrophotometer and the quality by nano-microfluidic capillary electrophoresis technology provided by Agilent 2100 Bioanalyzer. We evaluated the amplification status of three housekeeping genes and one small nuclear RNA gene using the ViiA 7 platform, with specific primers. Results Every step of the sample handling protocol, which begins with sample harvest and ends with the data analysis, is of utmost importance due to the fact that it is time consuming, labor intensive, and highly expensive. High temperature of the surgical procedure does not affect the small nucleic acid sequences in comparison with the mRNA. Conclusion Gene expression is clearly affected by the RNA quality, but less affected in the case of small nuclear RNAs. We proved that the high-temperature, highly invasive transurethral resection of bladder tumor procedure damages the tissue and affects the integrity of the RNA from biological specimens. PMID:27330317

The Model-Based Study of the Effectiveness of Reporting Lists of Small Feature Sets Using RNA-Seq Data.

PubMed

Kim, Eunji; Ivanov, Ivan; Hua, Jianping; Lampe, Johanna W; Hullar, Meredith Aj; Chapkin, Robert S; Dougherty, Edward R

2017-01-01

Ranking feature sets for phenotype classification based on gene expression is a challenging issue in cancer bioinformatics. When the number of samples is small, all feature selection algorithms are known to be unreliable, producing significant error, and error estimators suffer from different degrees of imprecision. The problem is compounded by the fact that the accuracy of classification depends on the manner in which the phenomena are transformed into data by the measurement technology. Because next-generation sequencing technologies amount to a nonlinear transformation of the actual gene or RNA concentrations, they can potentially produce less discriminative data relative to the actual gene expression levels. In this study, we compare the performance of ranking feature sets derived from a model of RNA-Seq data with that of a multivariate normal model of gene concentrations using 3 measures: (1) ranking power, (2) length of extensions, and (3) Bayes features. This is the model-based study to examine the effectiveness of reporting lists of small feature sets using RNA-Seq data and the effects of different model parameters and error estimators. The results demonstrate that the general trends of the parameter effects on the ranking power of the underlying gene concentrations are preserved in the RNA-Seq data, whereas the power of finding a good feature set becomes weaker when gene concentrations are transformed by the sequencing machine.

Smoking, Sex, and Non-Small Cell Lung Cancer: Steroid Hormone Receptors in Tumor Tissue (S0424).

PubMed

Cheng, Ting-Yuan David; Darke, Amy K; Redman, Mary W; Zirpoli, Gary R; Davis, Warren; Payne Ondracek, Rochelle; Bshara, Wiam; Omilian, Angela R; Kratzke, Robert; Reid, Mary E; Molina, Julian R; Kolesar, Jill M; Chen, Yuhchyau; MacRae, Robert M; Moon, James; Mack, Philip; Gandara, David R; Kelly, Karen; Santella, Regina M; Albain, Kathy S; Ambrosone, Christine B

2018-01-13

To what extent steroid hormones contribute to lung cancer in male and female never smokers and smokers is unclear. We examined expression of hormone receptors in lung tumors by sex and smoking. Patients with primary non-small cell lung cancer were recruited into an Intergroup study in the United States and Canada, led by SWOG (S0424). Tumors from 813 cases (450 women and 363 men) were assayed using immunohistochemistry for estrogen receptor (ER)-α, ER-β, progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Linear regression was used to examine differences in expression by sex and smoking status. Cox proportional hazard models were used to estimate survival associated with the receptors. All statistical tests were two-sided. In ever smokers, postmenopause and oral contraceptive use were associated with lower nuclear ER-β (P = .02) and total (nuclear + cytoplasmic) PR expression (P = .02), respectively. Women had lower cytoplasmic ER-α (regression coefficient [β], or differences in H-scores = -15.8, P = .003) and nuclear ER-β (β = -12.8, P = .04) expression than men, adjusting for age, race, and smoking. Ever smokers had both higher cytoplasmic ER-α (β = 45.0, P < .001) and ER-β (β = 25.9, P < .001) but lower total PR (β = -42.1, P < .001) than never smokers. Higher cytoplasmic ER-α and ER-β were associated with worse survival (hazard ratio = 1.73, 95% confidence interval [CI] = 1.15 to 2.58, and HR = 1.59, 95% CI = 1.08 to 2.33, respectively; quartiles 4 vs 1). Lower expression of nuclear ER-β in women supports the estrogen hypothesis in lung cancer etiology. Increasing cytoplasmic ER-α and ER-β and decreasing PR protein expression may be mechanisms whereby smoking disrupts hormone pathways. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

A comparison of the male customers of female street prostitutes with national samples of men.

PubMed

Monto, Martin A; McRee, Nick

2005-10-01

Previous research on customers of prostitutes has relied on small samples and qualitative interviews. Conceptions of customers have tended toward either the "every man" perspective, which implies customers are no different than other men, or the "peculiar man" perspective, which implies customers are characterized by stark differences or psychological inadequacies. This study compares a large sample of men (N = 1672) arrested for trying to hire street prostitutes with nationally representative samples of men. Customers were less likely to be married, less likely to be happily married if married, and more likely to report being unhappy in general than men in the national samples. Customers also expressed greater sexual liberalism and reported thinking about sex, masturbating, and participating in other aspects of the sex industry more frequently than men in general. Most differences were small, indicating customers as a category differ from other men in degree rather than quality.

General Relativistic Precession in Small Solar System Bodies

NASA Astrophysics Data System (ADS)

Sekhar, Aswin; Werner, Stephanie; Hoffmann, Volker; Asher, David; Vaubaillon, Jeremie; Hajdukova, Maria; Li, Gongjie

2016-10-01

Introduction: One of the greatest successes of the Einstein's General Theory of Relativity (GR) was the correct prediction of the precession of perihelion of Mercury. The closed form expression to compute this precession tells us that substantial GR precession would occur only if the bodies have a combination of both moderately small perihelion distance and semi-major axis. Minimum Orbit Intersection Distance (MOID) is a quantity which helps us to understand the closest proximity of two orbits in space. Hence evaluating MOID is crucial to understand close encounters and collision scenarios better. In this work, we look at the possible scenarios where a small GR precession in argument of pericentre (ω) can create substantial changes in MOID for small bodies ranging from meteoroids to comets and asteroids.Analytical Approach and Numerical Integrations: Previous works have looked into neat analytical techniques to understand different collision scenarios and we use those standard expressions to compute MOID analytically. We find the nature of this mathematical function is such that a relatively small GR precession can lead to drastic changes in MOID values depending on the initial value of ω. Numerical integrations were done with package MERCURY incorporating the GR code to test the same effects. Numerical approach showed the same interesting relationship (as shown by analytical theory) between values of ω and the peaks/dips in MOID values. Previous works have shown that GR precession suppresses Kozai oscillations and this aspect was verified using our integrations. There is an overall agreement between both analytical and numerical methods.Summary and Discussion: We find that GR precession could play an important role in the calculations pertaining to MOID and close encounter scenarios in the case of certain small solar system bodies (depending on their initial orbital elements). Previous works have looked into impact probabilities and collision scenarios on planets from different small body populations. This work aims to find certain sub-sets of orbits where GR could play an interesting role. Certain parallels are drawn between the cases of asteroids, comets and small perihelion distance meteoroid streams.

Correlation between molecular analysis, diagnosis according to the 2015 WHO classification of unresected lung tumours and TTF1 expression in small biopsies and cytology specimens from 344 non-small cell lung carcinoma patients.

PubMed

Russell, Prudence A; Rogers, Toni-Maree; Solomon, Benjamin; Alam, Naveed; Barnett, Stephen A; Rathi, Vivek; Williams, Richard A; Wright, Gavin M; Conron, Matthew

2017-10-01

We investigated correlations between diagnosis according to the 2015 World Health Organization (WHO) classification of unresected lung tumours, molecular analysis and TTF1 expression in small biopsy and cytology specimens from 344 non-small cell lung carcinoma (NSCLC) patients. One case failed testing for EGFR, KRAS and ALK abnormalities and six had insufficient tumour for ALK testing. Overall mutation rate in 343 cases was 48% for the genes tested, with 19% EGFR, 33% KRAS and 4% BRAF mutations, and 5% ALK rearrangements detected. More EGFR-mutant (78%) and ALK-rearranged (75%) tumours had morphologic adenocarcinoma than KRAS-mutant (56%) tumours. Despite no significant difference in the overall rate of any molecular abnormality between morphologic adenocarcinoma (52%) and NSCLC, favour adenocarcinoma (47%) (p = 0.18), KRAS mutations were detected more frequently in the latter group. No significant difference in the overall rate of any molecular abnormality between TTF1 positive (49%) and TTF1 negative tumours (44%) (p = 0.92) was detected, but more EGFR-mutant (97%) and ALK-rearranged tumours (92%) were TTF1 positive than KRAS-mutant tumours (68%). Rates of EGFR, KRAS and BRAF mutations and ALK rearrangements in this Australian NSCLC patient population are consistent with the published international literature. Our findings suggest that 2015 WHO classification of unresected tumours may assist in identifying molecular subsets of advanced NSCLC. Copyright © 2017 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.

Identification of small RNAs in extracellular vesicles from the commensal yeast Malassezia sympodialis.

PubMed

Rayner, Simon; Bruhn, Sören; Vallhov, Helen; Andersson, Anna; Billmyre, R Blake; Scheynius, Annika

2017-01-04

Malassezia is the dominant fungus in the human skin mycobiome and is associated with common skin disorders including atopic eczema (AE)/dermatitis. Recently, it was found that Malassezia sympodialis secretes nanosized exosome-like vesicles, designated MalaEx, that carry allergens and can induce inflammatory cytokine responses. Extracellular vesicles from different cell-types including fungi have been found to deliver functional RNAs to recipient cells. In this study we assessed the presence of small RNAs in MalaEx and addressed if the levels of these RNAs differ when M. sympodialis is cultured at normal human skin pH versus the elevated pH present on the skin of patients with AE. The total number and the protein concentration of the released MalaEx harvested after 48 h culture did not differ significantly between the two pH conditions nor did the size of the vesicles. From small RNA sequence data, we identified a set of reads with well-defined start and stop positions, in a length range of 16 to 22 nucleotides consistently present in the MalaEx. The levels of small RNAs were not significantly differentially expressed between the two different pH conditions indicating that they are not influenced by the elevated pH level observed on the AE skin.

Aggressive rat prostate tumors reprogram the benign parts of the prostate and regional lymph nodes prior to metastasis

PubMed Central

Thysell, Elin; Halin Bergström, Sofia; Bergh, Anders

2017-01-01

In order to grow and spread tumors need to interact with adjacent tissues. We therefore hypothesized that small but aggressive prostate cancers influence the rest of the prostate and regional lymph nodes differently than tumors that are more indolent. Poorly metastatic (Dunning AT1) or highly metastatic (Dunning MLL) rat prostate tumor cells were injected into the ventral prostate lobe of immunocompetent rats. After 10 days—when the tumors occupied about 30% of the prostate lobe and lymph node metastases were undetectable—the global gene expression in tumors, benign parts of the prostate, and regional iliac lymph nodes were examined to define tumor-induced changes related to preparation for future metastasis. The tumors induced profound effects on the gene expression profiles in the benign parts of the prostate and these were strikingly different in the two tumor models. Gene ontology enrichment analysis suggested that tumors with high metastatic capacity were more successful than less metastatic tumors in inducing tumor-promoting changes and suppressing anti-tumor immune responses in the entire prostate. Some of these differences such as altered angiogenesis, nerve density, accumulation of T-cells and macrophages were verified by immunohistochemistry. Gene expression alterations in the regional lymph nodes suggested decreased quantity and activation of immune cells in MLL-lymph nodes that were also verified by immunostaining. In summary, even when small highly metastatic prostate tumors can affect the entire tumor-bearing organ and pre-metastatic lymph nodes differently than less metastatic tumors. When the kinetics of these extratumoral influences (by us named TINT = tumor instructed normal tissue) are more precisely defined they could potentially be used as markers of disease aggressiveness and become therapeutic targets. PMID:28472073

MicroRNA-dependent regulation of transcription in non-small cell lung cancer.

PubMed

Molina-Pinelo, Sonia; Gutiérrez, Gabriel; Pastor, Maria Dolores; Hergueta, Marta; Moreno-Bueno, Gema; García-Carbonero, Rocío; Nogal, Ana; Suárez, Rocío; Salinas, Ana; Pozo-Rodríguez, Francisco; Lopez-Rios, Fernando; Agulló-Ortuño, Maria Teresa; Ferrer, Irene; Perpiñá, Asunción; Palacios, José; Carnero, Amancio; Paz-Ares, Luis

2014-01-01

Squamous cell lung cancer (SCC) and adenocarcinoma are the most common histological subtypes of non-small cell lung cancer (NSCLC), and have been traditionally managed in the clinic as a single entity. Increasing evidence, however, illustrates the biological diversity of these two histological subgroups of lung cancer, and supports the need to improve our understanding of the molecular basis beyond the different phenotypes if we aim to develop more specific and individualized targeted therapy. The purpose of this study was to identify microRNA (miRNA)-dependent transcriptional regulation differences between SCC and adenocarcinoma histological lung cancer subtypes. In this work, paired miRNA (667 miRNAs by TaqMan Low Density Arrays (TLDA)) and mRNA profiling (Whole Genome 44 K array G112A, Agilent) was performed in tumor samples of 44 NSCLC patients. Nine miRNAs and 56 mRNAs were found to be differentially expressed in SCC versus adenocarcinoma samples. Eleven of these 56 mRNA were predicted as targets of the miRNAs identified to be differently expressed in these two histological conditions. Of them, 6 miRNAs (miR-149, miR-205, miR-375, miR-378, miR-422a and miR-708) and 9 target genes (CEACAM6, CGN, CLDN3, ABCC3, MLPH, ACSL5, TMEM45B, MUC1) were validated by quantitative PCR in an independent cohort of 41 lung cancer patients. Furthermore, the inverse correlation between mRNAs and microRNAs expression was also validated. These results suggest miRNA-dependent transcriptional regulation differences play an important role in determining key hallmarks of NSCLC, and may provide new biomarkers for personalized treatment strategies.

MicroRNA-Dependent Regulation of Transcription in Non-Small Cell Lung Cancer

PubMed Central

Molina-Pinelo, Sonia; Gutiérrez, Gabriel; Pastor, Maria Dolores; Hergueta, Marta; Moreno-Bueno, Gema; García-Carbonero, Rocío; Nogal, Ana; Suárez, Rocío; Salinas, Ana; Pozo-Rodríguez, Francisco; Lopez-Rios, Fernando; Agulló-Ortuño, Maria Teresa; Ferrer, Irene; Perpiñá, Asunción; Palacios, José; Carnero, Amancio; Paz-Ares, Luis

2014-01-01

Squamous cell lung cancer (SCC) and adenocarcinoma are the most common histological subtypes of non-small cell lung cancer (NSCLC), and have been traditionally managed in the clinic as a single entity. Increasing evidence, however, illustrates the biological diversity of these two histological subgroups of lung cancer, and supports the need to improve our understanding of the molecular basis beyond the different phenotypes if we aim to develop more specific and individualized targeted therapy. The purpose of this study was to identify microRNA (miRNA)-dependent transcriptional regulation differences between SCC and adenocarcinoma histological lung cancer subtypes. In this work, paired miRNA (667 miRNAs by TaqMan Low Density Arrays (TLDA)) and mRNA profiling (Whole Genome 44 K array G112A, Agilent) was performed in tumor samples of 44 NSCLC patients. Nine miRNAs and 56 mRNAs were found to be differentially expressed in SCC versus adenocarcinoma samples. Eleven of these 56 mRNA were predicted as targets of the miRNAs identified to be differently expressed in these two histological conditions. Of them, 6 miRNAs (miR-149, miR-205, miR-375, miR-378, miR-422a and miR-708) and 9 target genes (CEACAM6, CGN, CLDN3, ABCC3, MLPH, ACSL5, TMEM45B, MUC1) were validated by quantitative PCR in an independent cohort of 41 lung cancer patients. Furthermore, the inverse correlation between mRNAs and microRNAs expression was also validated. These results suggest miRNA-dependent transcriptional regulation differences play an important role in determining key hallmarks of NSCLC, and may provide new biomarkers for personalized treatment strategies. PMID:24625834

Ubiquitin over-expression phenotypes and ubiquitin gene molecular misreading during aging in Drosophila melanogaster

PubMed Central

Hoe, Nicholas; Huang, Chung M.; Landis, Gary; Verhage, Marian; Ford, Daniel; Yang, Junsheng; van Leeuwen, Fred W.; Tower, John

2011-01-01

Molecular Misreading (MM) is the inaccurate conversion of genomic information into aberrant proteins. For example, when RNA polymerase II transcribes a GAGAG motif it synthesizes at low frequency RNA with a two-base deletion. If the deletion occurs in a coding region, translation will result in production of misframed proteins. During mammalian aging, misframed versions of human amyloid precursor protein (hApp) and ubiquitin (hUbb) accumulate in the aggregates characteristic of neurodegenerative diseases, suggesting dysfunctional degradation or clearance. Here cDNA clones encoding wild-type hUbb and the frame-shifted version hUbb+1 were expressed in transgenic Drosophila using the doxycycline-regulated system. Misframed proteins were abundantly produced, both from the transgenes and from endogenous Drosophila ubiquitin-encoding genes, and their abundance increased during aging in whole-fly extracts. Over-expression of wild-type hUbb, but not hUbb+1, was toxic during fly development. In contrast, when over-expressed specifically in adult flies, hUbb+1 caused small decreases in life span, whereas hUbb was associated with small increases, preferentially in males. The data suggest that MM occurs in Drosophila and that the resultant misframed proteins accumulate with age. MM of the ubiquitin gene can produce alternative ubiquitin gene products with different and sometimes opposing phenotypic effects. PMID:21415465

Identification of differentially expressed genes associated with differential body size in mandarin fish (Siniperca chuatsi).

PubMed

Tian, Changxu; Li, Ling; Liang, Xu-Fang; He, Shan; Guo, Wenjie; Lv, Liyuan; Wang, Qingchao; Song, Yi

2016-08-01

Body size is an obvious and important characteristic of fish. Mandarin fish Siniperca chuatsi (Basilewsky) is one of the most valuable perciform species widely cultured in China. Individual differences in body size are common in mandarin fish and significantly influence the aquaculture production. However, little is currently known about its genetic control. In this study, digital gene expression profiling and transcriptome sequencing were performed in mandarin fish with differential body size at 30 and 180 days post-hatch (dph), respectively. Body weight, total length and body length of fish with big-size were significantly higher than those with small-size at both 30 and 180 dph (P < 0.05). 2171 and 2014 differentially expressed genes were identified between small-size and big-size fish at 30 and 180 dph, respectively. RT quantitative PCR (qPCR) analysis showed that the differential expression of 10 selected genes in mandarin fish that went through the same training procedure. The genes were involved in the growth hormone-insulin-like growth factor axis, cell proliferation and differentiation, appetite control, glucose metabolism, reproduction and sexual size dimorphism pathways. This study will help toward a comprehensive understanding of the complexity of regulation of body size in mandarin fish individuals and provide valuable information for future research.

Systematic approach identifies RHOA as a potential biomarker therapeutic target for Asian gastric cancer.

PubMed

Chang, Hae Ryung; Nam, Seungyoon; Lee, Jinhyuk; Kim, Jin-Hee; Jung, Hae Rim; Park, Hee Seo; Park, Sungjin; Ahn, Young Zoo; Huh, Iksoo; Balch, Curt; Ku, Ja-Lok; Powis, Garth; Park, Taesung; Jeong, Jin-Hyun; Kim, Yon Hui

2016-12-06

Gastric cancer (GC) is a highly heterogeneous disease, in dire need of specific, biomarker-driven cancer therapies. While the accumulation of cancer "Big Data" has propelled the search for novel molecular targets for GC, its specific subpathway and cellular functions vary from patient to patient. In particular, mutations in the small GTPase gene RHOA have been identified in recent genome-wide sequencing of GC tumors. Moreover, protein overexpression of RHOA was reported in Chinese populations, while RHOA mutations were found in Caucasian GC tumors. To develop evidence-based precision medicine for heterogeneous cancers, we established a systematic approach to integrate transcriptomic and genomic data. Predicted signaling subpathways were then laboratory-validated both in vitro and in vivo, resulting in the identification of new candidate therapeutic targets. Here, we show: i) differences in RHOA expression patterns, and its pathway activity, between Asian and Caucasian GC tumors; ii) in vitro and in vivo perturbed RHOA expression inhibits GC cell growth in high RHOA-expressing cell lines; iii) inverse correlation between RHOA and RHOB expression; and iv) an innovative small molecule design strategy for RHOA inhibitors. In summary, RHOA, and its oncogenic signaling pathway, represent a strong biomarker-driven therapeutic target for Asian GC. This comprehensive strategy represents a promising approach for the development of "hit" compounds.

[Effect of gene optimization on the expression and purification of HDV small antigen produced by genetic engineering].

PubMed

Ding, Jun-Ying; Meng, Qing-Ling; Guo, Min-Zhuo; Yi, Yao; Su, Qiu-Dong; Lu, Xue-Xin; Qiu, Feng; Bi, Sheng-Li

2012-10-01

To study the effect of gene optimization on the expression and purification of HDV small antigen produced by genetic engineering. Based on the colon preference of E. coli, the HDV small antigen original gene from GenBank was optimized. Both the original gene and the optimized gene expressed in prokaryotic cells, SDS-PAGE was made to analyze the protein expression yield and to decide which protein expression style was more proportion than the other. Furthermore, two antigens were purified by chromatography in order to compare the purity by SDS-PAGE and Image Lab software. SDS-PAGE indicated that the molecular weight of target proteins from two groups were the same as we expected. Gene optimization resulted in the higher yield and it could make the product more soluble. After chromatography, the purity of target protein from optimized gene was up to 96.3%, obviously purer than that from original gene. Gene optimization could increase the protein expression yield and solubility of genetic engineering HDV small antigen. In addition, the product from the optimized gene group was easier to be purified for diagnosis usage.

Lactobacillus gasseri in the Upper Small Intestine Impacts an ACSL3-Dependent Fatty Acid-Sensing Pathway Regulating Whole-Body Glucose Homeostasis.

PubMed

Bauer, Paige V; Duca, Frank A; Waise, T M Zaved; Dranse, Helen J; Rasmussen, Brittany A; Puri, Akshita; Rasti, Mozhgan; O'Brien, Catherine A; Lam, Tony K T

2018-03-06

Long-chain acyl-CoA synthetase (ACSL)-dependent upper small intestinal lipid metabolism activates pre-absorptive pathways to regulate metabolic homeostasis, but whether changes in the upper small intestinal microbiota alter specific fatty acid-dependent pathways to impact glucose homeostasis remains unknown. We here first find that upper small intestinal infusion of Intralipid, oleic acid, or linoleic acid pre-absorptively increases glucose tolerance and lowers glucose production in rodents. High-fat feeding impairs pre-absorptive fatty acid sensing and reduces upper small intestinal Lactobacillus gasseri levels and ACSL3 expression. Transplantation of healthy upper small intestinal microbiota to high-fat-fed rodents restores L. gasseri levels and fatty acid sensing via increased ACSL3 expression, while L. gasseri probiotic administration to non-transplanted high-fat-fed rodents is sufficient to restore upper small intestinal ACSL3 expression and fatty acid sensing. In summary, we unveil a glucoregulatory role of upper small intestinal L. gasseri that impacts an ACSL3-dependent glucoregulatory fatty acid-sensing pathway. Copyright © 2018 Elsevier Inc. All rights reserved.

Fluorescence diffuse tomography for detection of RFP-expressed tumors in small animals

NASA Astrophysics Data System (ADS)

Turchin, Ilya V.; Savitsky, Alexander P.; Kamensky, Vladislav A.; Plehanov, Vladimir I.; Orlova, Anna G.; Kleshnin, Mikhail S.; Shirmanova, Marina V.; Fix, Ilya I.; Popov, Vladimir O.

2007-07-01

Capabilities of tumor detection by different optical methods can be significantly improved by labeling of tumors with fluorescent markers. Creation of tumor cell lines transfected with fluorescent proteins provides the possibility not only to detect tumor, but also to conduct the intravital monitoring studies. Cell lines of human melanomas Mel-P, Mel-Kor and human embryonic kidney HEK-293 Phoenix were transfected with DsRed-Express and Turbo-RFP genes. Emission of RFP in the long-wave optical range permits detection of the deeply located tumors, which is essential for whole-body imaging. Only special tools for turbid media imaging, such as fluorescent diffusion tomography (FDT), enable noninvasive investigation of the internal structure of biological tissue. FDT setup for monitoring of tumor growth in small animals has been created. An animal is scanned in the transilluminative configuration by low-frequency modulated light (1 kHz) from Nd:YAG laser with second harmonic generation at the 532 nm wavelength. An optimizing algorithm for scanning of an experimantal animal is suggested. In vivo experiments were conducted immediately after the subcutaneously injection of fluorescing cells into small animals. It was shown that FDT method allows to detect the presence of fluorescent cells in small animals and can be used for monitoring of tumor growth and anticancer drug responce.

Teachers' Stories of Change: Stress, Care and Economic Rationality

ERIC Educational Resources Information Center

Easthope, Chris; Easthope, Gary

2007-01-01

The impact of economic rationalism on teachers' working lives has been documented extensively, particularly in the UK. This article provides a case study of its impact in the early 1990s in a small Australian state, Tasmania, to illustrate that although the particular institutional forms through which it is expressed may differ its impact is…

Different Anaphoric Expressions Are Investigated by Event-Related Brain Potentials

ERIC Educational Resources Information Center

Streb, Judith; Hennighausen, Erwin; Rosler, Frank

2004-01-01

Event-related potentials were recorded to substantiate the claim of a distinct psycholinguistic status of (a) pronouns vs. proper names and (b) ellipses vs. proper names. In two studies 41 students read sentences in which the number of intervening words between the anaphor and its antecedent was either small or large. Comparing the far with the…

Expression of GULP1 in bronchial epithelium is associated with the progression of emphysema in chronic obstructive pulmonary disease.

PubMed

Datta, Sayantan; Nam, Hae-Seong; Hayashi, Masamichi; Maldonado, Leonel; Goldberg, Rachel; Brait, Mariana; Sidransky, David; Illei, Peter; Baras, Alex; Vij, Neeraj; Hoque, Mohammad O

2017-03-01

GULP1 functions as a cytoplasmic adaptor protein involved in the engulfment of apoptotic cells. The aim of this study was to investigate the expression and/or promoter methylation of GULP1 in the bronchial tissue and the lung parenchyma of chronic obstructive pulmonary disease (COPD) patients and control subjects without COPD (non-smokers and smokers). Using a case-control design, we selected a group of 15 subjects with non-small cell lung carcinoma (NSCLC), 15 subjects with COPD, 9 subjects of without COPD (4 non-smokers and 5 smokers) as controls. We studied the expression of GULP1 in the bronchial tissue and the lung parenchyma by means of immunohistochemistry (IHC). To understand the mechanistic aspect of GULP1 expression in COPD and NSCLC, we performed quantitative methylation specific PCR (QMSP) in cases and controls of COPD and NSCLC. Our IHC analysis revealed that GULP1 was not expressed in pneumocytes or alveolar macrophages of COPD patients, however, GULP1 expression was detected at different levels in bronchial epithelium. GULP1 expression statistically correlated with severity of COPD-emphysema (p = 0.001, χ 2 test). GULP1 promoter methylation was not observed by QMSP assay in any of the samples thereby excluding the role of promoter methylation in differential expression of GULP1 in COPD and NSCLC. This study provides preliminary observations on the differences in GULP1 expression in different cellular components of lung tissues from COPD and control subjects. Our findings suggest a potential role for GULP1 in the pathogenesis and progression of COPD-emphysema that warrants further investigation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Emotion-independent face recognition

NASA Astrophysics Data System (ADS)

De Silva, Liyanage C.; Esther, Kho G. P.

2000-12-01

Current face recognition techniques tend to work well when recognizing faces under small variations in lighting, facial expression and pose, but deteriorate under more extreme conditions. In this paper, a face recognition system to recognize faces of known individuals, despite variations in facial expression due to different emotions, is developed. The eigenface approach is used for feature extraction. Classification methods include Euclidean distance, back propagation neural network and generalized regression neural network. These methods yield 100% recognition accuracy when the training database is representative, containing one image representing the peak expression for each emotion of each person apart from the neutral expression. The feature vectors used for comparison in the Euclidean distance method and for training the neural network must be all the feature vectors of the training set. These results are obtained for a face database consisting of only four persons.

An Update on Cellular MicroRNA Expression in Human Papillomavirus-Associated Head and Neck Squamous Cell Carcinoma.

PubMed

Emmett, Sarah; Whiteman, David C; Panizza, Benedict J; Antonsson, Annika

2018-06-19

Squamous cell carcinoma of mucosal sites in the head and neck (HNSCC) is the sixth most common cause of cancer worldwide, and despite advances in conventional management, it still has significant morbidity and mortality associated with both diagnosis and treatment. Advances in our understanding of the biological mechanisms underlying this disease have demonstrated a significant difference between human papillomavirus (HPV)-associated, HPV and tobacco associated, and HPV-negative disease. It remains important to further elucidate the biologic and genetic differences between HPV-associated and tobacco-associated disease, with the aim of earlier diagnosis through screening, and advances in management including the development of novel therapeutic agents. MicroRNAs (miRNAs) are small, non-coding RNAs that function as post-transcriptional regulators of gene expression, and have effects on almost every cellular function, and have potentially important applications to diagnosis, management and prognosis in HNSCC. Establishing a cellular miRNA expression profile for HPV-associated disease may therefore have important implications for the screening and treatment of this disease. This review summarises the current findings regarding miRNA expression in mucosal HNSCC, and focuses particularly on miRNA expression in HPV-associated tumours. © 2018 S. Karger AG, Basel.

Gene Expression Profiling of Transcription Factors of Helicobacter pylori under Different Environmental Conditions.

PubMed

De la Cruz, Miguel A; Ares, Miguel A; von Bargen, Kristine; Panunzi, Leonardo G; Martínez-Cruz, Jessica; Valdez-Salazar, Hilda A; Jiménez-Galicia, César; Torres, Javier

2017-01-01

Helicobacter pylori is a Gram-negative bacterium that colonizes the human gastric mucosa and causes peptic ulcers and gastric carcinoma. H. pylori strain 26695 has a small genome (1.67 Mb), which codes for few known transcriptional regulators that control bacterial metabolism and virulence. We analyzed by qRT-PCR the expression of 16 transcriptional regulators in H. pylori 26695, including the three sigma factors under different environmental conditions. When bacteria were exposed to acidic pH, urea, nickel, or iron, the sigma factors were differentially expressed with a particularly strong induction of fliA . The regulatory genes hrcA, hup , and crdR were highly induced in the presence of urea, nickel, and iron. In terms of biofilm formation fliA, flgR, hp1021, fur, nikR , and crdR were induced in sessile bacteria. Transcriptional expression levels of rpoD, flgR, hspR, hp1043 , and cheY were increased in contact with AGS epithelial cells. Kanamycin, chloramphenicol, and tetracycline increased or decreased expression of regulatory genes, showing that these antibiotics affect the transcription of H. pylori . Our data indicate that environmental cues which may be present in the human stomach modulate H. pylori transcription.

Rebamipide inhibits indomethacin-induced small intestinal injury: possible involvement of intestinal microbiota modulation by upregulation of α-defensin 5.

PubMed

Tanigawa, Tetsuya; Watanabe, Toshio; Otani, Koji; Nadatani, Yuji; Ohkawa, Fumikazu; Sogawa, Mitsue; Yamagami, Hirokazu; Shiba, Masatsugu; Watanabe, Kenji; Tominaga, Kazunari; Fujiwara, Yasuhiro; Takeuchi, Koji; Arakawa, Tetsuo

2013-03-15

Enterobacteria play important roles in the pathophysiology of small intestinal injuries induced by nonsteroidal anti-inflammatory drugs (NSAIDs). We investigated the effects of rebamipide, a gastrointestinal mucoprotective drug, on indomethacin-induced small intestinal injuries, intestinal microbiota, and expression levels of α-defensin 5, which is a Paneth cell-specific antimicrobial peptide and is important for the regulation of intestinal microbiota. Indomethacin (10mg/kg) was orally administered to mice after oral administration of rebamipide (100 or 300 mg/kg) or vehicle for 1 week, and the small intestinal injuries were assessed. After oral administration of rebamipide, the small intestinal contents were subjected to terminal restriction fragment length polymorphism (T-RFLP) analysis to assess the intestinal microbiota composition. Further, the expression levels of mRNA and protein for α-defensin 5 in the ileal tissue were determined by real-time reverse transcription-polymerase chain reaction and western blotting analysis, respectively. Rebamipide inhibited indomethacin-induced small intestinal injuries and T-RFLP analysis showed that rebamipide increased the percentage of Lactobacillales and decreased the percentage of Bacteroides and Clostridium than that in vehicle-treated controls. The mice that were treated with rebamipide showed an increase in α-defensin 5 mRNA expression and protein levels in the ileal tissue compared to vehicle-treated control mice. Indomethacin reduced expression of α-defensin 5 mRNA in ileal tissue, while rebamipide reversed expression of α-defensin 5 mRNA. In conclusion, our study results suggest that rebamipide inhibits indomethacin-induced small intestinal injuries, possibly by modulating microbiota in the small intestine by upregulation of α-defensin 5. Copyright © 2013 Elsevier B.V. All rights reserved.

Identification and comparative analysis of drought-associated microRNAs in two cowpea genotypes.

PubMed

Barrera-Figueroa, Blanca E; Gao, Lei; Diop, Ndeye N; Wu, Zhigang; Ehlers, Jeffrey D; Roberts, Philip A; Close, Timothy J; Zhu, Jian-Kang; Liu, Renyi

2011-09-17

Cowpea (Vigna unguiculata) is an important crop in arid and semi-arid regions and is a good model for studying drought tolerance. MicroRNAs (miRNAs) are known to play critical roles in plant stress responses, but drought-associated miRNAs have not been identified in cowpea. In addition, it is not understood how miRNAs might contribute to different capacities of drought tolerance in different cowpea genotypes. We generated deep sequencing small RNA reads from two cowpea genotypes (CB46, drought-sensitive, and IT93K503-1, drought-tolerant) that grew under well-watered and drought stress conditions. We mapped small RNA reads to cowpea genomic sequences and identified 157 miRNA genes that belong to 89 families. Among 44 drought-associated miRNAs, 30 were upregulated in drought condition and 14 were downregulated. Although miRNA expression was in general consistent in two genotypes, we found that nine miRNAs were predominantly or exclusively expressed in one of the two genotypes and that 11 miRNAs were drought-regulated in only one genotype, but not the other. These results suggest that miRNAs may play important roles in drought tolerance in cowpea and may be a key factor in determining the level of drought tolerance in different cowpea genotypes.

Blood-gene expression reveals reduced circadian rhythmicity in individuals resistant to sleep deprivation.

PubMed

Arnardottir, Erna S; Nikonova, Elena V; Shockley, Keith R; Podtelezhnikov, Alexei A; Anafi, Ron C; Tanis, Keith Q; Maislin, Greg; Stone, David J; Renger, John J; Winrow, Christopher J; Pack, Allan I

2014-10-01

To address whether changes in gene expression in blood cells with sleep loss are different in individuals resistant and sensitive to sleep deprivation. Blood draws every 4 h during a 3-day study: 24-h normal baseline, 38 h of continuous wakefulness and subsequent recovery sleep, for a total of 19 time-points per subject, with every 2-h psychomotor vigilance task (PVT) assessment when awake. Sleep laboratory. Fourteen subjects who were previously identified as behaviorally resistant (n = 7) or sensitive (n = 7) to sleep deprivation by PVT. Thirty-eight hours of continuous wakefulness. We found 4,481 unique genes with a significant 24-h diurnal rhythm during a normal sleep-wake cycle in blood (false discovery rate [FDR] < 5%). Biological pathways were enriched for biosynthetic processes during sleep. After accounting for circadian effects, two genes (SREBF1 and CPT1A, both involved in lipid metabolism) exhibited small, but significant, linear changes in expression with the duration of sleep deprivation (FDR < 5%). The main change with sleep deprivation was a reduction in the amplitude of the diurnal rhythm of expression of normally cycling probe sets. This reduction was noticeably higher in behaviorally resistant subjects than sensitive subjects, at any given P value. Furthermore, blood cell type enrichment analysis showed that the expression pattern difference between sensitive and resistant subjects is mainly found in cells of myeloid origin, such as monocytes. Individual differences in behavioral effects of sleep deprivation are associated with differences in diurnal amplitude of gene expression for genes that show circadian rhythmicity. © 2014 Associated Professional Sleep Societies, LLC.

The miRNA Transcriptome Directly Reflects the Physiological and Biochemical Differences between Red, White, and Intermediate Muscle Fiber Types

PubMed Central

Ma, Jideng; Wang, Hongmei; Liu, Rui; Jin, Long; Tang, Qianzi; Wang, Xun; Jiang, Anan; Hu, Yaodong; Li, Zongwen; Zhu, Li; Li, Ruiqiang; Li, Mingzhou; Li, Xuewei

2015-01-01

MicroRNAs (miRNAs) are small non-coding RNAs that can regulate their target genes at the post-transcriptional level. Skeletal muscle comprises different fiber types that can be broadly classified as red, intermediate, and white. Recently, a set of miRNAs was found expressed in a fiber type-specific manner in red and white fiber types. However, an in-depth analysis of the miRNA transcriptome differences between all three fiber types has not been undertaken. Herein, we collected 15 porcine skeletal muscles from different anatomical locations, which were then clearly divided into red, white, and intermediate fiber type based on the ratios of myosin heavy chain isoforms. We further illustrated that three muscles, which typically represented each muscle fiber type (i.e., red: peroneal longus (PL), intermediate: psoas major muscle (PMM), white: longissimus dorsi muscle (LDM)), have distinct metabolic patterns of mitochondrial and glycolytic enzyme levels. Furthermore, we constructed small RNA libraries for PL, PMM, and LDM using a deep sequencing approach. Results showed that the differentially expressed miRNAs were mainly enriched in PL and played a vital role in myogenesis and energy metabolism. Overall, this comprehensive analysis will contribute to a better understanding of the miRNA regulatory mechanism that achieves the phenotypic diversity of skeletal muscles. PMID:25938964

Differential Expression of MicroRNAs in Breast Cancers from Four Different Ethnicities.

PubMed

Pollard, Jennifer; Burns, Phil A; Hughes, Tom A; Ho-Yen, Colan; Jones, J Louise; Mukherjee, Geetashree; Omoniyi-Esan, Ganiat O; Titloye, Nicholas Akinwale; Speirs, Valerie; Shaaban, Abeer M

2018-05-23

Breast cancer outcomes vary across different ethnic groups. MicroRNAs (miRs) are small non-coding RNA molecules that regulate gene expression across a range of pathologies, including breast cancer. The aim of this study was to evaluate the presence and expression of miRs in breast cancer samples from different ethnic groups. Breast cancer tissue from 4 ethnic groups, i.e., British Caucasian, British Black, Nigerian, and Indian, were identified and matched for patients' age, tumour grade/type, and 10 × 10 µm sections taken. Tumour areas were macrodissected, total RNA was extracted, and cDNA was synthesised. cDNA was applied to human miScript PCR arrays allowing the quantification of 84 of the most abundantly expressed/best-characterised miRs. Differential expression of 9 miRs was seen across the 4 groups. Significantly higher levels of miR-140-5p, miR-194 and miR-423-5p (the last of which harbours the single-nucleotide polymorphism rs6505162) were seen in the breast tumours of Nigerian patients when compared with other ethnic groups (all p < 0.0001). miR-101 was overexpressed in breast cancers in the Indian patients. An in silico analysis of miR-423-5p showed that the AC genotype is mainly associated with Europeans (57%), while Asians display mostly CC (approx. 60%), and Africans mainly AA (approx. 60%). This study shows divergence in miR expression in breast cancers from different ethnic groups, and suggests that specific genetic variants in miR genes may affect breast cancer risk in these groups. Predicted targets of these miRs may uncover useful biomarkers that could have clinical value in breast cancers in different ethnic groups. © 2018 S. Karger AG, Basel.

Express Payload Project - A new method for rapid access to Space Station Freedom

NASA Technical Reports Server (NTRS)

Uhran, Mark L.; Timm, Marc G.

1993-01-01

The deployment and permanent operation of Space Station Freedom will enable researchers to enter a new era in the 21st century, in which continuous on-orbit experimentation and observation become routine. In support of this objective, the Space Station Freedom Program Office has initiated the Express Payload Project. The fundamental project goal is to reduce the marginal cost associated with small payload development, integration, and operation. This is to be accomplished by developing small payload accommodations hardware and a new streamlined small payload integration process. Standardization of small payload interfaces, certification of small payload containers, and increased payload developer responsibility for mission success are key aspects of the Express Payload Project. As the project progresses, the principles will be applied to both pressurized payloads flown inside the station laboratories and unpressurized payloads attached to the station external structures. The increased access to space afforded by Space Station Freedom and the Express Payload Project has the potential to significantly expand the scope, magnitude, and success of future research in the microgravity environment.

[Mechanism of Chlorogenic Acid in Apoptotic Regulation through Notch1 
Pathway in Non-small Cell Lung Carcinoma in Animal Level].

PubMed

Li, Wei; Liu, Xu; Zhang, Guoqian; Zhang, Linlin

2017-08-20

It has been proven that chlorogenic acids can produce anticancer effects by regulating cell cycle, inducing apoptosis, inhibiting cell growth, Notch signaling pathways are closely related to many human tumors. The aim of this study is to study the mechanism of chlorogenic acid on apoptosis of non-small lung cancer through Notch1 pathway in animal level, and hope to provide theory basis on clinical treatment and research aimed at targeting Notch1 signaling in non-small cell carcinoma (NSCLC). MTT assay was used to evaluate the A549 cell proliferation under the treatment of chlorogenic acid. The effect of chlorogenic acid on apoptotic and cell cycle were detected by flow cytometry. The animal model of A549 cell transplanted in nude was established, tumer size and weight were detected. The mRNA level of Notch1 signal pathway related facter were detected by RT-PCR; the expression of Notch1 signal pathway related facter in tumor tissue was detected by western blot. Chlorogenic acid inhibited the A549 cell proliferation. incresed cell apoptotic and cell percentagein G2/M (P<0.05), and in a dose-dependent manner. In animal model, tumer size and weight were lower than control group, the difference was statistically significant (P<0.05). The relative expression of mRNA of Notch1, VEGF, Delta4, HES1 and HEY1 were decreaced (P<0.05) in tumor tissue which treated with chlorogenic. The expression of Notch1 were decreaced, PTEN, p-PTEN, p-AKT were increced significantly in tumor tissue which treated with chlorogenic (P<0.05). Chlorogenic acid can regulate theapoptosis of non-small lung cancer through Notch pathway in animal level, which may be associated with the down-regulating the expression of VEGF and Delta4. Notch pathway may cross talk with PI3K/AKT pathway through PTEN in NSCLC.

Development and application of a quantitative multiplexed small GTPase activity assay using targeted proteomics.

PubMed

Zhang, Cheng-Cheng; Li, Ru; Jiang, Honghui; Lin, Shujun; Rogalski, Jason C; Liu, Kate; Kast, Juergen

2015-02-06

Small GTPases are a family of key signaling molecules that are ubiquitously expressed in various types of cells. Their activity is often analyzed by western blot, which is limited by its multiplexing capability, the quality of isoform-specific antibodies, and the accuracy of quantification. To overcome these issues, a quantitative multiplexed small GTPase activity assay has been developed. Using four different binding domains, this assay allows the binding of up to 12 active small GTPase isoforms simultaneously in a single experiment. To accurately quantify the closely related small GTPase isoforms, a targeted proteomic approach, i.e., selected/multiple reaction monitoring, was developed, and its functionality and reproducibility were validated. This assay was successfully applied to human platelets and revealed time-resolved coactivation of multiple small GTPase isoforms in response to agonists and differential activation of these isoforms in response to inhibitor treatment. This widely applicable approach can be used for signaling pathway studies and inhibitor screening in many cellular systems.

Effects of aspirin on small-cell lung cancer mortality and metastatic presentation.

PubMed

Maddison, Paul

2017-04-01

Although meta-analysis data have shown that taking regular aspirin may reduce lung cancer mortality, individual trial data results are conflicting, and the data on the effects of aspirin on different histological subtypes of lung tumours, in particular small-cell lung cancer, are sparse. We conducted a prospective observational study of 313 patients with a new diagnosis of small-cell lung cancer and recorded use of aspirin before and after tumour diagnosis. Seventy-one (23%) patients were taking regular daily aspirin for more than 2 years at the time of tumour diagnosis. We found that regular use of aspirin had no effect on survival nor metastatic presentation compared to data from small-cell lung cancer patients not taking aspirin. The lack of survival benefit in patients with small-cell lung cancer taking long-term aspirin may be due to the low expression of cyclooxygenase-2 in small-cell lung cancer tissue. Copyright © 2017 Elsevier B.V. All rights reserved.

Facial expression identification using 3D geometric features from Microsoft Kinect device

NASA Astrophysics Data System (ADS)

Han, Dongxu; Al Jawad, Naseer; Du, Hongbo

2016-05-01

Facial expression identification is an important part of face recognition and closely related to emotion detection from face images. Various solutions have been proposed in the past using different types of cameras and features. Microsoft Kinect device has been widely used for multimedia interactions. More recently, the device has been increasingly deployed for supporting scientific investigations. This paper explores the effectiveness of using the device in identifying emotional facial expressions such as surprise, smile, sad, etc. and evaluates the usefulness of 3D data points on a face mesh structure obtained from the Kinect device. We present a distance-based geometric feature component that is derived from the distances between points on the face mesh and selected reference points in a single frame. The feature components extracted across a sequence of frames starting and ending by neutral emotion represent a whole expression. The feature vector eliminates the need for complex face orientation correction, simplifying the feature extraction process and making it more efficient. We applied the kNN classifier that exploits a feature component based similarity measure following the principle of dynamic time warping to determine the closest neighbors. Preliminary tests on a small scale database of different facial expressions show promises of the newly developed features and the usefulness of the Kinect device in facial expression identification.

On the theory of the relativistic motion of a charged particle in the field of intense electromagnetic radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Milant'ev, V. P., E-mail: vmilantiev@sci.pfu.edu.ru; Castillo, A. J., E-mail: vmilant@mail.ru

2013-04-15

Averaged relativistic equations of motion of a charged particle in the field of intense electromagnetic radiation have been obtained in the geometrical optics approximation using the Bogoliubov method. Constraints are determined under which these equations are valid. Oscillating additions to the smoothed dynamical variables of the particle have been found; they are reduced to known expressions in the case of the circularly and linearly polarized plane waves. It has been shown that the expressions for the averaged relativistic force in both cases contain new additional small terms weakening its action. The known difference between the expressions for the ponderomotive forcemore » in the cases of circularly and linearly polarized waves has been confirmed.« less

Association of mRNA expression of iron metabolism-associated genes and progression of non-alcoholic steatohepatitis in rats.

PubMed

Higuchi, Teruhisa; Moriyama, Mitsuhiko; Fukushima, Akiko; Matsumura, Hiroshi; Matsuoka, Shunichi; Kanda, Tatsuo; Sugitani, Masahiko; Tsunemi, Akiko; Ueno, Takahiro; Fukuda, Noboru

2018-05-25

Excess iron is associated with non-alcoholic steatohepatitis (NASH). mRNA expression of duodenal cytochrome b, divalent metal transporter 1, ferroportin 1, hepcidin, hephaestin and transferrin receptor 1 in liver were higher in high fat, high cholesterol-containing diet (HFCD) group than in normal diet (ND) group. mRNA levels of divalent metal transporter 1 and transferrin receptor 1, which stimulate iron absorption and excretion, were enhanced in small intestine. Epithelial mucosa of small intestine in HFCD group was characterized by plasma cell and eosinophil infiltration and increased vacuoles. Iron absorption was enhanced in this NASH model in the context of chronic inflammation of small intestinal epithelial cells, consequences of intestinal epithelial cell impairment caused by HFCD. Iron is transported to hepatocytes via portal blood, and abnormalities in iron absorption and excretion occur in small intestine from changes in iron transporter expression, which also occurs in NASH liver. Knockdown of hepcidin antimicrobial peptide led to enhanced heavy chain of ferritin expression in human hepatocytes, indicating association between hepcidin production and iron storage in hepatocytes. Iron-related transporters in liver and lower/upper portions of small intestine play critical roles in NASH development. Expression of iron metabolism-related genes in liver and small intestine was analyzed in stroke-prone spontaneously hypertensive rats (SHR-SP), which develop NASH. Five-week-old SHR-SP fed ND or HFCD were examined. mRNA and protein levels of iron metabolism-related genes in liver and small intestine from 12- and 19-week-old rats were evaluated by real-time RT-PCR and immunohistochemistry or Western blot.

Gluten affects epithelial differentiation-associated genes in small intestinal mucosa of coeliac patients

PubMed Central

Juuti-Uusitalo, K; Mäki, M; Kainulainen, H; Isola, J; Kaukinen, K

2007-01-01

In coeliac disease gluten induces an immunological reaction in genetically susceptible patients, and influences on epithelial cell proliferation and differentiation in the small-bowel mucosa. Our aim was to find novel genes which operate similarly in epithelial proliferation and differentiation in an epithelial cell differentiation model and in coeliac disease patient small-bowel mucosal biopsy samples. The combination of cDNA microarray data originating from a three-dimensional T84 epithelial cell differentiation model and small-bowel mucosal biopsy samples from untreated and treated coeliac disease patients and healthy controls resulted in 30 genes whose mRNA expression was similarly affected. Nine of 30 were located directly or indirectly in the receptor tyrosine kinase pathway starting from the epithelial growth factor receptor. Removal of gluten from the diet resulted in a reversion in the expression of 29 of the 30 genes in the small-bowel mucosal biopsy samples. Further characterization by blotting and labelling revealed increased epidermal growth factor receptor and beta-catenin protein expression in the small-bowel mucosal epithelium in untreated coeliac disease patients compared to healthy controls and treated coeliac patients. We found 30 genes whose mRNA expression was affected similarly in the epithelial cell differentiation model and in the coeliac disease patient small-bowel mucosal biopsy samples. In particular, those genes involved in the epithelial growth factor-mediated signalling pathways may be involved in epithelial cell differentiation and coeliac disease pathogenesis. The epithelial cell differentiation model is a useful tool for studying gene expression changes in the crypt–villus axis. PMID:17888028

In Silico Analysis of Small RNAs Suggest Roles for Novel and Conserved miRNAs in the Formation of Epigenetic Memory in Somatic Embryos of Norway Spruce.

PubMed

Yakovlev, Igor A; Fossdal, Carl G

2017-01-01

Epigenetic memory in Norway spruce affects the timing of bud burst and bud set, vitally important adaptive traits for this long-lived forest species. Epigenetic memory is established in response to the temperature conditions during embryogenesis. Somatic embryogenesis at different epitype inducing (EpI) temperatures closely mimics the natural processes of epigenetic memory formation in seeds, giving rise to epigenetically different clonal plants in a reproducible and predictable manner, with respect to altered bud phenology. MicroRNAs (miRNAs) and other small non-coding RNAs (sRNAs) play an essential role in the regulation of plant gene expression and may affect this epigenetic mechanism. We used NGS sequencing and computational in silico methods to identify and profile conserved and novel miRNAs among small RNAs in embryogenic tissues of Norway spruce at three EpI temperatures (18, 23 and 28°C). We detected three predominant classes of sRNAs related to a length of 24 nt, followed by a 21-22 nt class and a third 31 nt class of sRNAs. More than 2100 different miRNAs within the prevailing length 21-22 nt were identified. Profiling these putative miRNAs allowed identification of 1053 highly expressed miRNAs, including 523 conserved and 530 novels. 654 of these miRNAs were found to be differentially expressed (DEM) depending on EpI temperature. For most DEMs, we defined their putative mRNA targets. The targets represented mostly by transcripts of multiple-repeats proteins, like TIR, NBS-LRR, PPR and TPR repeat, Clathrin/VPS proteins, Myb-like, AP2, etc. Notably, 124 DE miRNAs targeted 203 differentially expressed epigenetic regulators. Developing Norway spruce embryos possess a more complex sRNA structure than that reported for somatic tissues. A variety of the predicted miRNAs showed distinct EpI temperature dependent expression patterns. These putative EpI miRNAs target spruce genes with a wide range of functions, including genes known to be involved in epigenetic regulation, which in turn could provide a feedback process leading to the formation of epigenetic marks. We suggest that TIR, NBS and LRR domain containing proteins could fulfill more general functions for signal transduction from external environmental stimuli and conversion them into molecular response. Fine-tuning of the miRNA production likely participates in both developmental regulation and epigenetic memory formation in Norway spruce.

Correlating chemical sensitivity and basal gene expression reveals mechanism of action | Office of Cancer Genomics

Cancer.gov

Changes in cellular gene expression in response to small-molecule or genetic perturbations have yielded signatures that can connect unknown mechanisms of action (MoA) to ones previously established. We hypothesized that differential basal gene expression could be correlated with patterns of small-molecule sensitivity across many cell lines to illuminate the actions of compounds whose MoA are unknown.

Lectin Ulex europaeus agglutinin I specifically labels a subset of primary afferent fibers which project selectively to the superficial dorsal horn of the spinal cord.

PubMed

Mori, K

1986-02-19

To examine differential carbohydrate expression among different subsets of primary afferent fibers, several fluorescein-isothiocyanate conjugated lectins were used in a histochemical study of the dorsal root ganglion (DRG) and spinal cord of the rabbit. The lectin Ulex europaeus agglutinin I specifically labeled a subset of DRG cells and primary afferent fibers which projected to the superficial laminae of the dorsal horn. These results suggest that specific carbohydrates containing L-fucosyl residue is expressed selectively in small diameter primary afferent fibers which subserve nociception or thermoception.

Drug-loaded nanoparticles induce gene expression in human pluripotent stem cell derivatives

NASA Astrophysics Data System (ADS)

Gajbhiye, Virendra; Escalante, Leah; Chen, Guojun; Laperle, Alex; Zheng, Qifeng; Steyer, Benjamin; Gong, Shaoqin; Saha, Krishanu

2013-12-01

Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained higher fibroblast proliferation levels and MMP activity. The results demonstrate that the PEG-H40-DXC nanoparticle system provides an effective tool to controlling gene expression in human stem cell derivatives.Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained higher fibroblast proliferation levels and MMP activity. The results demonstrate that the PEG-H40-DXC nanoparticle system provides an effective tool to controlling gene expression in human stem cell derivatives. Electronic supplementary information (ESI) available: ESI containing 1H NMR spectra and additional fibroblast characterization data. See DOI: 10.1039/c3nr04794f

Validating internal controls for quantitative plant gene expression studies

PubMed Central

Brunner, Amy M; Yakovlev, Igor A; Strauss, Steven H

2004-01-01

Background Real-time reverse transcription PCR (RT-PCR) has greatly improved the ease and sensitivity of quantitative gene expression studies. However, accurate measurement of gene expression with this method relies on the choice of a valid reference for data normalization. Studies rarely verify that gene expression levels for reference genes are adequately consistent among the samples used, nor compare alternative genes to assess which are most reliable for the experimental conditions analyzed. Results Using real-time RT-PCR to study the expression of 10 poplar (genus Populus) housekeeping genes, we demonstrate a simple method for determining the degree of stability of gene expression over a set of experimental conditions. Based on a traditional method for analyzing the stability of varieties in plant breeding, it defines measures of gene expression stability from analysis of variance (ANOVA) and linear regression. We found that the potential internal control genes differed widely in their expression stability over the different tissues, developmental stages and environmental conditions studied. Conclusion Our results support that quantitative comparisons of candidate reference genes are an important part of real-time RT-PCR studies that seek to precisely evaluate variation in gene expression. The method we demonstrated facilitates statistical and graphical evaluation of gene expression stability. Selection of the best reference gene for a given set of experimental conditions should enable detection of biologically significant changes in gene expression that are too small to be revealed by less precise methods, or when highly variable reference genes are unknowingly used in real-time RT-PCR experiments. PMID:15317655

Differential expression patterns of housekeeping genes increase diagnostic and prognostic value in lung cancer

PubMed Central

Chang, Yu-Chun; Ding, Yan; Dong, Lingsheng; Zhu, Lang-Jing; Jensen, Roderick V.

2018-01-01

Background Using DNA microarrays, we previously identified 451 genes expressed in 19 different human tissues. Although ubiquitously expressed, the variable expression patterns of these “housekeeping genes” (HKGs) could separate one normal human tissue type from another. Current focus on identifying “specific disease markers” is problematic as single gene expression in a given sample represents the specific cellular states of the sample at the time of collection. In this study, we examine the diagnostic and prognostic potential of the variable expressions of HKGs in lung cancers. Methods Microarray and RNA-seq data for normal lungs, lung adenocarcinomas (AD), squamous cell carcinomas of the lung (SQCLC), and small cell carcinomas of the lung (SCLC) were collected from online databases. Using 374 of 451 HKGs, differentially expressed genes between pairs of sample types were determined via two-sided, homoscedastic t-test. Principal component analysis and hierarchical clustering classified normal lung and lung cancers subtypes according to relative gene expression variations. We used uni- and multi-variate cox-regressions to identify significant predictors of overall survival in AD patients. Classifying genes were selected using a set of training samples and then validated using an independent test set. Gene Ontology was examined by PANTHER. Results This study showed that the differential expression patterns of 242, 245, and 99 HKGs were able to distinguish normal lung from AD, SCLC, and SQCLC, respectively. From these, 70 HKGs were common across the three lung cancer subtypes. These HKGs have low expression variation compared to current lung cancer markers (e.g., EGFR, KRAS) and were involved in the most common biological processes (e.g., metabolism, stress response). In addition, the expression pattern of 106 HKGs alone was a significant classifier of AD versus SQCLC. We further highlighted that a panel of 13 HKGs was an independent predictor of overall survival and cumulative risk in AD patients. Discussion Here we report HKG expression patterns may be an effective tool for evaluation of lung cancer states. For example, the differential expression pattern of 70 HKGs alone can separate normal lung tissue from various lung cancers while a panel of 106 HKGs was a capable class predictor of subtypes of non-small cell carcinomas. We also reported that HKGs have significantly lower variance compared to traditional cancer markers across samples, highlighting the robustness of a panel of genes over any one specific biomarker. Using RNA-seq data, we showed that the expression pattern of 13 HKGs is a significant, independent predictor of overall survival for AD patients. This reinforces the predictive power of a HKG panel across different gene expression measurement platforms. Thus, we propose the expression patterns of HKGs alone may be sufficient for the diagnosis and prognosis of individuals with lung cancer. PMID:29761043

Limiting cases of the small-angle scattering approximation solutions for the propagation of laser beams in anisotropic scattering media

NASA Technical Reports Server (NTRS)

Box, M. A.; Deepak, A.

1981-01-01

The propagation of photons in a medium with strongly anisotropic scattering is a problem with a considerable history. Like the propagation of electrons in metal foils, it may be solved in the small-angle scattering approximation by the use of Fourier-transform techniques. In certain limiting cases, one may even obtain analytic expressions. This paper presents some of these results in a model-independent form and also illustrates them by the use of four different phase-function models. Sample calculations are provided for comparison purposes

Dimensional crossover in fragmentation

NASA Astrophysics Data System (ADS)

Sotolongo-Costa, Oscar; Rodriguez, Arezky H.; Rodgers, G. J.

2000-11-01

Experiments in which thick clay plates and glass rods are fractured have revealed different behavior of fragment mass distribution function in the small and large fragment regions. In this paper we explain this behavior using non-extensive Tsallis statistics and show how the crossover between the two regions is caused by the change in the fragments’ dimensionality during the fracture process. We obtain a physical criterion for the position of this crossover and an expression for the change in the power-law exponent between the small and large fragment regions. These predictions are in good agreement with the experiments on thick clay plates.

The significance of PIWI family expression in human lung embryogenesis and non-small cell lung cancer.

PubMed

Navarro, Alfons; Tejero, Rut; Viñolas, Nuria; Cordeiro, Anna; Marrades, Ramon M; Fuster, Dolors; Caritg, Oriol; Moises, Jorge; Muñoz, Carmen; Molins, Laureano; Ramirez, Josep; Monzo, Mariano

2015-10-13

The expression of Piwi-interacting RNAs, small RNAs that bind to PIWI proteins, was until recently believed to be limited to germinal stem cells. We have studied the expression of PIWI genes during human lung embryogenesis and in paired tumor and normal tissue prospectively collected from 71 resected non-small-cell lung cancer patients. The mRNA expression analysis showed that PIWIL1 was highly expressed in 7-week embryos and downregulated during the subsequent weeks of development. PIWIL1 was expressed in 11 of the tumor samples but in none of the normal tissue samples. These results were validated by immunohistochemistry, showing faint cytoplasmic reactivity in the PIWIL1-positive samples. Interestingly, the patients expressing PIWIL1 had a shorter time to relapse (TTR) (p = 0.006) and overall survival (OS) (p = 0.0076) than those without PIWIL1 expression. PIWIL2 and 4 were downregulated in tumor tissue in comparison to the normal tissue (p < 0.001) and the patients with lower levels of PIWIL4 had shorter TTR (p = 0.048) and OS (p = 0.033). In the multivariate analysis, PIWIL1 expression emerged as an independent prognostic marker. Using 5-Aza-dC treatment and bisulfite sequencing, we observed that PIWIL1 expression could be regulated in part by methylation. Finally, an in silico study identified a stem-cell expression signature associated with PIWIL1 expression.

Prognostic value of the MicroRNA regulators Dicer and Drosha in non-small-cell lung cancer: co-expression of Drosha and miR-126 predicts poor survival.

PubMed

Lønvik, Kenneth; Sørbye, Sveinung W; Nilsen, Marit N; Paulssen, Ruth H

2014-01-01

Dicer and Drosha are important enzymes for processing microRNAs. Recent studies have exhibited possible links between expression of different miRNAs, levels of miRNA processing enzymes, and cancer prognosis. We have investigated the prognostic impact of Dicer and Drosha and their correlation with miR-126 expression in a large cohort of non-small cell lung cancer (NSCLC) patients. We aimed to find patient groups within the cohort that might have an advantage of receiving adjunctive therapies. Dicer expression in the cytoplasm and Drosha expression in the nucleus were evaluated by manual immunohistochemistry of tissue microarrays (TMAs), including tumor tissue samples from 335 patients with resected stages I to IIIA NSCLC. In addition, in situ hybridizations of TMAs for visualization of miR-126 were performed. Kaplan-Meier analysis was performed, and the log-rank test via SPSS v.22 was used for estimating significance levels. In patients with normal performance status (ECOG = 0, n = 197), high Dicer expression entailed a significantly better prognosis than low Dicer expression (P = 0.024). Dicer had no significant prognostic value in patients with reduced performance status (ECOG = 1-2, n = 138). High Drosha expression was significantly correlated with high levels of the microRNA 126 (miR-126) (P = 0.004). Drosha/miR-126 co-expression had a significant negative impact on the disease-specific survival (DSS) rate (P < 0.001). Multivariate analyses revealed that the interaction Dicer*Histology (P = 0.049) and Drosha/miR-126 co-expression (P = 0.033) were independent prognostic factors. In NSCLC patients with normal performance status, Dicer is a positive prognostic factor. The importance of Drosha as a prognostic factor in our material seems to be related to miR-126 and possibly other microRNAs.

Two different protein expression profiles of oral squamous cell carcinoma analyzed by immunoprecipitation high-performance liquid chromatography.

PubMed

Kim, Soung Min; Jeong, Dasul; Kim, Min Keun; Lee, Sang Shin; Lee, Suk Keun

2017-08-08

Oral squamous cell carcinoma (OSCC) is one of the most dangerous cancers in the body, producing serious complications with individual behaviors. Many different pathogenetic factors are involved in the carcinogenesis of OSCC. Cancer cells derived from oral keratinocytes can produce different carcinogenic signaling pathways through differences in protein expression, but their protein expression profiles cannot be easily explored with ordinary detection methods. The present study compared the protein expression profiles between two different types of OSCCs, which were analyzed through immunoprecipitation high-performance liquid chromatography (IP-HPLC). Two types of squamous cell carcinoma (SCC) occurred in a mandibular (SCC-1) and maxillary gingiva (SCC-2), but their clinical features and progression were quite different from each other. SCC-1 showed a large gingival ulceration with severe halitosis and extensive bony destruction, while SCC-2 showed a relatively small papillary gingival swelling but rapidly grew to form a large submucosal mass, followed by early cervical lymph node metastasis. In the histological observation, SCC-1 was relatively well differentiated with a severe inflammatory reaction, while SCC-2 showed severely infiltrative growth of each cancer islets accompanied with a mild inflammatory reaction. IP-HPLC analysis revealed contrary protein expression profiles analyzed by 72 different oncogenic proteins. SCC-1 showed more cellular apoptosis and invasive growth than SCC-2 through increased expression of caspases, MMPs, p53 signaling, FAS signaling, TGF-β1 signaling, and angiogenesis factors, while SCC-2 showed more cellular growth and survival than SCC-1 through the increased expression of proliferating factors, RAS signaling, eIF5A signaling, WNT signaling, and survivin. The increased trends of cellular apoptosis and invasiveness in the protein expression profiles of SCC-1 were implicative of its extensive gingival ulceration and bony destruction, while the increased trends of cellular proliferation and survival in the protein profile of SCC-2 were implicative of its rapid growing tumor mass and early lymph node metastasis. These analyses of the essential oncogenic protein expression profiles in OSCC provide important information for genetic counseling or customized gene therapy in cancer treatment. Therefore, protein expression profile analysis through IP-HPLC is helpful not only for the molecular genetic diagnosis of cancer but also in identifying target molecules for customized gene therapy in near future.

What do somatic hypermutation and class switch recombination teach us about chronic lymphocytic leukaemia pathogenesis?

PubMed

Oppezzo, P; Dighiero, G

2005-01-01

B-CLL cells express CD5 and IgM/IgD and thus have a mantle zone-like phenotype of naive cells, which, in normal conditions express unmutated Ig genes. However, recent studies have shown that 50%-70% of CLL harbour somatic mutations of VH genes, as if they had matured in a lymphoid follicle. Interestingly, the presence or absence of somatic hypermutation (SHM) process is associated with the use of particular VH genes. Particular alleles of the VH1-69 gene and the VH4-39 gene are preferentially expressed in an unmutated form, while VH4-34 or the majority of VH3 family genes frequently contain somatic mutations. The fact that some genes like VH1-69 and VH3-07 recombine this VH segment to particular JH segments and the restricted use of CDR3 sequences by CLLs expressing the VH4-39 gene suggest that the observed differences in BCR structure in B-CLL could result from selection by distinct antigenic epitopes. It is currently unclear whether this putative antigen-driven process could occur prior to leukaemic transformation and/or that the precursors were transformed into leukaemic cells at distinct maturational stages. The mutational profile of Ig genes has been shown to be associated with disease prognosis. These results could favour the idea that CLL could correspond to two different diseases that look alike in morphologic and phenotypic terms. In CLL with mutated Ig genes, the proliferating B cell may have transited through germinal centres, the physiologic site of hypermutation, whereas in CLL with unmutated Ig genes the malignant B cell may derive from a pre-germinal centre naïve B cell. Despite these clinical and molecular differences, recent studies on gene expression profiling of B-CLL cells showed that CLL is characterized by a common gene expression signature that is irrespective of Ig mutational status and differs from other lymphoid cancers and normal lymphoid subpopulations, suggesting that CLL cases share a common mechanism of transformation and/or cell of origin. Activation induced cytidine deaminase (AID) plays a key role in SHM and class switch recombination (CSR). However, the mechanisms accounting for AID action and control of its expression remain unclear. In a recent work we have shown that in contrast to normal circulating B-cells, AID transcripts are expressed constitutively in CLL patients undergoing active CSR, but interestingly this expression occurs predominately in unmutated CLL B-cells. These data favour the view that AID protein may act differentially on CSR and SHM pathways, but the role-played by AID in both processes remains to be elucidated. Recent work indicates that AID is expressed in a small fraction of tumoral cells, which could suggest that this small fraction of cells may correspond to B-CLL cells that would have recently experienced an AID-inducing stimulus occurring in a specific microenvironment.

Synergistic Effect of Auto-Activation and Small RNA Regulation on Gene Expression

NASA Astrophysics Data System (ADS)

Xiong, Li-Ping; Ma, Yu-Qiang; Tang, Lei-Han

2010-09-01

Auto-activation and small ribonucleic acid (RNA)-mediated regulation are two important mechanisms in controlling gene expression. We study the synergistic effect of these two regulations on gene expression. It is found that under this combinatorial regulation, gene expression exhibits bistable behaviors at the transition regime, while each of these two regulations, if working solely, only leads to monostability. Within the stochastic framework, the base pairing strength between sRNA and mRNA plays an important role in controlling the transition time between on and off states. The noise strength of protein number in the off state approaches 1 and is smaller than that in the on state. The noise strength also depends on which parameters, the feedback strength or the synthesis rate of small RNA, are tuned in switching the gene expression on and off. Our findings may provide a new insight into gene-regulation mechanism and can be applied in synthetic biology.

[Expression profiles of the exosomal miRNAs in the chronic hepatitis B patients with persistently normal ALT].

PubMed

Li, Ronghua; Fu, Xiaoyu; Tang, Yujing; Fu, Lei; Tan, Deming; Ouyang, Yi; Peng, Shifang

2018-05-28

To investigate expression profiles of the plasma exosomal miRNAs of the chronic hepatitis B (CHB) patients with persistently normal alamine aminotransferase (PNALT) for the first time and try to find exosomal miRNAs which could reflect liver inflammation better. 
 Methods: Five CHB patients with liver tissue inflammation grade ≥A2 of PNALT and 5 CHB patients with liver tissue inflammation grade

Immunosuppressive therapies after intestinal transplant modulate the expression of Th1 signature genes during acute cellular rejection. Implications in the search for rejection biomarkers.

PubMed

Zambernardi, Agustina; Chiodetti, Ana; Meier, Dominik; Cabanne, Ana; Nachman, Fabio; Solar, Héctor; Rumbo, Carolina; Gondolesi, Gabriel E; Rumbo, Martin

2014-12-01

Acute cellular rejection (ACR) and infections are leading causes of graft loss and death in intestinal transplant patients. Our aim was to evaluate the impact of maintenance immunosuppressive therapies on the expression of pro-inflammatory mediators in small bowel at ACR diagnosis. We analyzed expression levels of Th1-associated genes, IFNG, CXCL10, and CXCL11 by qPCR in 46 selected graft biopsies unequivocally assigned to mild ACR (n = 14) or normal histopathology and clinical condition (n = 32) from 15 patients receiving two different immunosuppressive (IS) schemes. Double treatment: corticosteroids and tacrolimus (n = 17) and triple treatment: sirolimus or mycophenolate mofetil in addition to the basal therapy (n = 29). IFNG, CXCL10, and CXCL11 were induced during rejection (p < 0.05; p < 0.005, and p < 0.05, respectively). However, when rejection and control groups were classified according to immunosuppressive treatment, in the rejection group, significant differences of IFNG, CXCL10, and CXCL11 expression (p < 0.001; p < 0.005, and 0.01, respectively) were detected, whereas no differences were observed in the control group. Gene expression of Th1 response mediators is higher during ACR. Triple IS group showed significantly lower expression of pro-inflammatory Th1 mediators during mild ACR indicating that use of these markers to monitor rejection can be affected by the IS treatment used. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Temporal Differences in MicroRNA Expression Patterns in Astrocytes and Neurons after Ischemic Injury

PubMed Central

Ziu, Mateo; Fletcher, Lauren; Rana, Shushan; Jimenez, David F.; Digicaylioglu, Murat

2011-01-01

MicroRNAs (miRNAs) are small, non-protein-coding RNA molecules that modulate gene translation. Their expression is altered in many central nervous system (CNS) injuries suggesting a role in the cellular response to stress. Current studies in brain tissue have not yet described the cell-specific temporal miRNA expression patterns following ischemic injury. In this study, we analyzed the expression alterations of a set of miRNAs in neurons and astrocytes subjected to 60 minutes of ischemia and collected at different time-points following this injury. To mimic ischemic conditions and reperfusion in vitro, cortical primary neuronal and astrocytic cultures prepared from fetal rats were first placed in oxygen and glucose deprived (OGD) medium for 60 minutes, followed by their transfer into normoxic pre-conditioned medium. Total RNA was extracted at different time-points after the termination of the ischemic insult and the expression levels of miRNAs were measured. In neurons exposed to OGD, expression of miR-29b was upregulated 2-fold within 6 h and up to 4-fold at 24 h post-OGD, whereas induction of miR-21 was upregulated 2-fold after 24 h when compared to expression in neurons under normoxic conditions. In contrast, in astrocytes, miR-29b and miR-21 were upregulated only after 12 h. MiR-30b, 107, and 137 showed expression alteration in astrocytes, but not in neurons. Furthermore, we show that expression of miR-29b was significantly decreased in neurons exposed to Insulin-Like Growth Factor I (IGF-I), a well documented neuroprotectant in ischemic models. Our study indicates that miRNAs expression is altered in neurons and astrocytes after ischemic injury. Furthermore, we found that following OGD, specific miRNAs have unique cell-specific temporal expression patterns in CNS. Therefore the specific role of each miRNA in different intracellular processes in ischemic brain and the relevance of their temporal and spatial expression patterns warrant further investigation that may lead to novel strategies for therapeutic interventions. PMID:21373187

Effect of correlated observation error on parameters, predictions, and uncertainty

USGS Publications Warehouse

Tiedeman, Claire; Green, Christopher T.

2013-01-01

Correlations among observation errors are typically omitted when calculating observation weights for model calibration by inverse methods. We explore the effects of omitting these correlations on estimates of parameters, predictions, and uncertainties. First, we develop a new analytical expression for the difference in parameter variance estimated with and without error correlations for a simple one-parameter two-observation inverse model. Results indicate that omitting error correlations from both the weight matrix and the variance calculation can either increase or decrease the parameter variance, depending on the values of error correlation (ρ) and the ratio of dimensionless scaled sensitivities (rdss). For small ρ, the difference in variance is always small, but for large ρ, the difference varies widely depending on the sign and magnitude of rdss. Next, we consider a groundwater reactive transport model of denitrification with four parameters and correlated geochemical observation errors that are computed by an error-propagation approach that is new for hydrogeologic studies. We compare parameter estimates, predictions, and uncertainties obtained with and without the error correlations. Omitting the correlations modestly to substantially changes parameter estimates, and causes both increases and decreases of parameter variances, consistent with the analytical expression. Differences in predictions for the models calibrated with and without error correlations can be greater than parameter differences when both are considered relative to their respective confidence intervals. These results indicate that including observation error correlations in weighting for nonlinear regression can have important effects on parameter estimates, predictions, and their respective uncertainties.

Expression of survivin and p53 in oral lichen planus, lichenoid reaction and lichenoid dysplasia: An immunohistochemical study

PubMed Central

Basheer, Shaini; Shameena, PM; Sudha, S; Varma, Sujatha; Vidyanath, S; Varekar, Aniruddha

2017-01-01

Context: The malignant transformation potential of oral lichen planus (OLP) and related lesions is a subject of great controversy. Aim: The aim of this study was to compare the expression of proteins related to apoptosis and tumour suppressor gene processes in OLP, oral lichenoid reaction (OLR) and oral lichenoid dysplasia (OLD). Materials and Methods The immunohistochemical study was carried out to investigate the expressions of survivin and p53 in a total of 30 lesional biopsy specimens - 10 cases each of OLP, OLR and OLD. The expression rates were further compared with 10 control specimens of normal oral mucosa (NORM). Results: Immunoreactivity for p53 was seen in 7 cases (70%) of OLD, 4 cases (40%) of OLP and 2 cases (20%) of OLR and none of NORM. We obtained a significant difference (P = 0.01) in mean p53 expression between the different entities. The positive staining rate of survivin was found to be significantly different between OLD (50%), OLP (10%), OLR (0%), and normal mucosa (0%) (P = 0.004). There was a positive correlation between p53 and survivin expression in OLP and OLD using Pearson's correlation coefficient. Conclusion: Lichenoid dysplasia has shown p53 and survivin expression in the range of not OLP, but leukoplakia. On the other hand, OLR seems to be an innocuous lesion. The study results with OLP are inconclusive but points toward a small but important malignant potential in OLP. This kind of comparative study highlights the importance of biopsying OLP and related lesions for proper diagnosis and appropriate management. PMID:29391729

Impact of a high-cholesterol diet on expression levels of Niemann-Pick C1-like 1 and intestinal transporters in rats and mice.

PubMed

Kawase, Atsushi; Araki, Yasuha; Ueda, Yukiko; Nakazaki, Sayaka; Iwaki, Masahiro

2016-08-01

Niemann-Pick C1-like 1 (NPC1L1), ATP-binding cassette (ABC)G5, and ABCG8 are all involved in intestinal cholesterol absorption. It is unclear whether a high-cholesterol (HC) diet affects the expression of these transporters in rats and mice as well as humans. We examined the effects of an HC diet on their expression in small intestine and the differences between rats and mice in the responsive of this expression to an HC diet. In addition to these transporters, alterations in six representative drug and nutrient transporters (multidrug resistance-associated protein, breast cancer resistance protein, peptide transporter, sodium-glucose linked transporter, glucose transporter, and L-type amino acid transporter) and transcriptional factors such as hepatocyte nuclear factor (HNF)4α, sterol regulatory element-binding protein (SREBP)2, and liver X receptor (LXR)α were determined. In rats and mice fed an HC diet for 7 days, the mRNA and protein levels of NPC1L1 in the small intestine were determined by real-time reverse transcription polymerase chain reaction and western blotting, respectively. The mRNA levels of ABCG5 and ABCG8, six representative transporters, and transcriptional factors such as HNF4α, SREBP2, and LXR were examined. Significant decreases in the expression levels of NPC1L1 were observed in mice, but not rats, fed the HC diet. The mRNA levels of ABCG5 and ABCG8 were significantly increased in HC rats but not in mice. Only minor changes in the mRNA levels of the other transporters were seen in HC rats and mice. Decreased mRNA levels of HNF4α and SREBP2 in mice could be involved in the reduction in NPC1L1 expression observed upon the introduction of an HC diet. These results indicate that the effects of an HC diet on the expression levels of NPC1L1, ABCG5, and ABCG8 differ between mice and rats.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cui Xiangshun; Li Xingyu; Kim, Nam-Hyung

To gain insights into the roles the paternal genome and chromosome number play in pre-implantation development, we cultured fertilized embryos and diploid and haploid parthenotes (DPs and HPs, respectively), and compared their development and gene expression patterns. The DPs and fertilized embryos did not differ in developmental ability but HPs development was slower and characterized by impaired compaction and blastocoel formation. Microarray analysis revealed that fertilized blastocysts expressed several genes at higher levels than DP blastocysts; these included the Y-chromosome-specific gene eukaryotic translation initiation factor 2, subunit 3, structural gene Y-linked (Eif2s3y) and the imprinting gene U2 small nuclear ribonucleoproteinmore » auxiliary factor 1, related sequence 1 (U2af1-rs1). We also found that when DPs and HPs were both harvested at 44 and 58 h of culture, they differed in the expression of 38 and 665 genes, respectively. However, when DPs and HPs were harvested at the midpoints of 4-cell stage (44 and 49 h, respectively), no differences in expression was observed. Similarly, when the DPs and HPs were harvested when they became blastocysts (102 and 138 h, respectively), only 15 genes showed disparate expression. These results suggest that while transcripts needed for early development are delayed in HPs, it does progress sufficiently for the generation of the various developmental stages despite the lack of genetic components.« less

Intratumoral gene expression of 5-fluorouracil pharmacokinetics-related enzymes in stage I and II non-small cell lung cancer patients treated with uracil-tegafur after surgery: a prospective multi-institutional study in Japan.

PubMed

Eguchi, Keisuke; Oyama, Takahiko; Tajima, Atsushi; Abiko, Tomohiro; Sawafuji, Makoto; Horio, Hirotoshi; Hashizume, Toshinori; Matsutani, Noriyuki; Kato, Ryoichi; Nakayama, Mitsuo; Kawamura, Masafumi; Kobayashi, Koichi

2015-01-01

This investigation was conducted to assess the use of the intratumoral mRNA expression levels of nucleic acid-metabolizing enzymes as biomarkers of adjuvant chemotherapy for non-small cell lung cancer (NSCLC) using uracil-tegafur in a multi-institutional prospective study. 236 patients with a completely resected NSCLC (adenocarcinoma and squamous cell carcinoma) of pathological stage IA (maximum tumor diameter of 2 cm or greater), IB, and II tumors were given a dose of 250 mg of uracil-tegafur per square meter of body surface area per day orally for two years after surgery. Intratumoral mRNA levels of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyltransferase (OPRT), and thymidine phosphorylase (TP) genes relative to an internal standard, β-actin, were determined using laser-capture microdissection and fluorescence-based real time PCR detection systems. Among 5-FU target enzymes, TS was the only one that showed a significant difference in the level of gene expression between the high and low gene expression groups, for both disease-free survival (DFS) and overall survival (OS), when patients were divided according to median values; 5-year DFS rates in high/low TS gene expression were 60.4% and 72.6%, respectively (p=0.050), 5-year OS rates were 78.1% and 88.6%, respectively (p=0.011). Cox's proportional hazard model indicated that the pathological stage and TS gene expression level were independent values for predicting DFS. The TS gene expression level was shown to be an independent predictive factor for DFS in stage I and II NSCLC patients who were treated with uracil-tegafur following surgery. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Discrimination of gender using facial image with expression change

NASA Astrophysics Data System (ADS)

Kuniyada, Jun; Fukuda, Takahiro; Terada, Kenji

2005-12-01

By carrying out marketing research, the managers of large-sized department stores or small convenience stores obtain the information such as ratio of men and women of visitors and an age group, and improve their management plan. However, these works are carried out in the manual operations, and it becomes a big burden to small stores. In this paper, the authors propose a method of men and women discrimination by extracting difference of the facial expression change from color facial images. Now, there are a lot of methods of the automatic recognition of the individual using a motion facial image or a still facial image in the field of image processing. However, it is very difficult to discriminate gender under the influence of the hairstyle and clothes, etc. Therefore, we propose the method which is not affected by personality such as size and position of facial parts by paying attention to a change of an expression. In this method, it is necessary to obtain two facial images with an expression and an expressionless. First, a region of facial surface and the regions of facial parts such as eyes, nose, and mouth are extracted in the facial image with color information of hue and saturation in HSV color system and emphasized edge information. Next, the features are extracted by calculating the rate of the change of each facial part generated by an expression change. In the last step, the values of those features are compared between the input data and the database, and the gender is discriminated. In this paper, it experimented for the laughing expression and smile expression, and good results were provided for discriminating gender.

Modulation of small intestinal phosphate transporter by dietary supplements of mineral phosphorus and phytase in broilers.

PubMed

Huber, Korinna; Zeller, Ellen; Rodehutscord, Markus

2015-05-01

Dietary phosphorus (P) is known as a main modulator of phosphate (Pi) transporter expression. The effect of supplemented mineral P with or without phytase on protein expression of two sodium-dependent Pi (NaPi) transporters and a calcium channel was studied in the small intestine of broilers. Thirty-six broilers were randomly assigned to six different diets at 15 days of age. Two levels of total P (tP, adjusted by monocalcium phosphate (MCP) supplementation), 0.39% (BD-) and 0.47% (BD+) were fed until day 25; and at each tP level, three levels of phytase were used with 0, 500, and 12,500 FTU/kg of an E. coli phytase. Mucosa samples from jejunum and ileum were taken and apical membranes were isolated by MgCl2 precipitation. Protein expression of NaPi IIb, NaPi type III (PiT1) and the calcium channel TRPV6 were semiquantitatively measured by Western blotting and jejunal mucosal phytase activity by measurement of Pi release. The jejunal NaPi IIb transporter was expressed with two distinct bands, which were modulated differently by diet. NaPi IIb Band1 increased (P < 0.05) and Band2 decreased (P < 0.05) with phytase supplementation but was not affected by MCP supplementation. This inverse modulation of Band1 and Band2 was significantly related to the amount of net absorbed P with higher expression of Band1 at higher amounts of net absorbed P. In addition, a second Pi transporter, PiT1, was detected in which ileal expression decreased (P < 0.05) in response to higher phytase supplementation. The expression of the calcium channel TRPV6 was increased in BD+ groups. A trend for an interaction between MCP and phytase supplementation on mucosal phytase activity was observed (P = 0.079) with a decrease in activity when BD+ with 12,500 FTU/kg phytase was fed. Chicken intestinal epithelial cells responded to dietary supplemented phytase and MCP by changing the Pi transporter expression in apical membranes. In conclusion, availability of Pi is most likely the key modulator of transporter protein expression. However, a contribution of lower inositol phosphates generated by phytases and other phosphatases may also be relevant. © 2015 Poultry Science Association Inc.

Euchromatic Transposon Insertions Trigger Production of Novel Pi- and Endo-siRNAs at the Target Sites in the Drosophila Germline

PubMed Central

Olovnikov, Ivan; Abramov, Yuri; Kalmykova, Alla

2014-01-01

The control of transposable element (TE) activity in germ cells provides genome integrity over generations. A distinct small RNA–mediated pathway utilizing Piwi-interacting RNAs (piRNAs) suppresses TE expression in gonads of metazoans. In the fly, primary piRNAs derive from so-called piRNA clusters, which are enriched in damaged repeated sequences. These piRNAs launch a cycle of TE and piRNA cluster transcript cleavages resulting in the amplification of piRNA and TE silencing. Using genome-wide comparison of TE insertions and ovarian small RNA libraries from two Drosophila strains, we found that individual TEs inserted into euchromatic loci form novel dual-stranded piRNA clusters. Formation of the piRNA-generating loci by active individual TEs provides a more potent silencing response to the TE expansion. Like all piRNA clusters, individual TEs are also capable of triggering the production of endogenous small interfering (endo-si) RNAs. Small RNA production by individual TEs spreads into the flanking genomic regions including coding cellular genes. We show that formation of TE-associated small RNA clusters can down-regulate expression of nearby genes in ovaries. Integration of TEs into the 3′ untranslated region of actively transcribed genes induces piRNA production towards the 3′-end of transcripts, causing the appearance of genic piRNA clusters, a phenomenon that has been reported in different organisms. These data suggest a significant role of TE-associated small RNAs in the evolution of regulatory networks in the germline. PMID:24516406

The small airway epithelium as a target for the adverse pulmonary effects of silver nanoparticle inhalation.

PubMed

Guo, Chang; Buckley, Alison; Marczylo, Tim; Seiffert, Joanna; Römer, Isabella; Warren, James; Hodgson, Alan; Chung, Kian Fan; Gant, Timothy W; Smith, Rachel; Leonard, Martin O

2018-05-11

Experimental modeling to identify specific inhalation hazards for nanomaterials has in the main focused on in vivo approaches. However, these models suffer from uncertainties surrounding species-specific differences and cellular targets for biologic response. In terms of pulmonary exposure, approaches which combine 'inhalation-like' nanoparticulate aerosol deposition with relevant human cell and tissue air-liquid interface cultures are considered an important complement to in vivo work. In this study, we utilized such a model system to build on previous results from in vivo exposures, which highlighted the small airway epithelium as a target for silver nanoparticle (AgNP) deposition. RNA-SEQ was used to characterize alterations in mRNA and miRNA within the lung. Organotypic-reconstituted 3D human primary small airway epithelial cell cultures (SmallAir) were exposed to the same spark-generated AgNP and at the same dose used in vivo, in an aerosol-exposure air-liquid interface (AE-ALI) system. Adverse effects were characterized using lactate, LDH release and alterations in mRNA and miRNA. Modest toxicological effects were paralleled by significant regulation in gene expression, reflective mainly of specific inflammatory events. Importantly, there was a level of concordance between gene expression changes observed in vitro and in vivo. We also observed a significant correlation between AgNP and mass equivalent silver ion (Ag + ) induced transcriptional changes in SmallAir cultures. In addition to key mechanistic information relevant for our understanding of the potential health risks associated with AgNP inhalation exposure, this work further highlights the small airway epithelium as an important target for adverse effects.

Hi-C Chromatin Interaction Networks Predict Co-expression in the Mouse Cortex

PubMed Central

Hulsman, Marc; Lelieveldt, Boudewijn P. F.; de Ridder, Jeroen; Reinders, Marcel

2015-01-01

The three dimensional conformation of the genome in the cell nucleus influences important biological processes such as gene expression regulation. Recent studies have shown a strong correlation between chromatin interactions and gene co-expression. However, predicting gene co-expression from frequent long-range chromatin interactions remains challenging. We address this by characterizing the topology of the cortical chromatin interaction network using scale-aware topological measures. We demonstrate that based on these characterizations it is possible to accurately predict spatial co-expression between genes in the mouse cortex. Consistent with previous findings, we find that the chromatin interaction profile of a gene-pair is a good predictor of their spatial co-expression. However, the accuracy of the prediction can be substantially improved when chromatin interactions are described using scale-aware topological measures of the multi-resolution chromatin interaction network. We conclude that, for co-expression prediction, it is necessary to take into account different levels of chromatin interactions ranging from direct interaction between genes (i.e. small-scale) to chromatin compartment interactions (i.e. large-scale). PMID:25965262

Small bowel carcinomas in celiac or Crohn's disease: distinctive histophenotypic, molecular and histogenetic patterns.

PubMed

Vanoli, Alessandro; Di Sabatino, Antonio; Martino, Michele; Klersy, Catherine; Grillo, Federica; Mescoli, Claudia; Nesi, Gabriella; Volta, Umberto; Fornino, Daniele; Luinetti, Ombretta; Fociani, Paolo; Villanacci, Vincenzo; D'Armiento, Francesco P; Cannizzaro, Renato; Latella, Giovanni; Ciacci, Carolina; Biancone, Livia; Paulli, Marco; Sessa, Fausto; Rugge, Massimo; Fiocca, Roberto; Corazza, Gino R; Solcia, Enrico

2017-10-01

Non-familial small bowel carcinomas are relatively rare and have a poor prognosis. Two small bowel carcinoma subsets may arise in distinct immune-inflammatory diseases (celiac disease and Crohn's disease) and have been recently suggested to differ in prognosis, celiac disease-associated carcinoma cases showing a better outcome, possibly due to their higher DNA microsatellite instability and tumor-infiltrating T lymphocytes. In this study, we investigated the histological structure (glandular vs diffuse/poorly cohesive, mixed or solid), cell phenotype (intestinal vs gastric/pancreatobiliary duct type) and Wnt signaling activation (β-catenin and/or SOX-9 nuclear expression) in a series of 26 celiac disease-associated small bowel carcinoma, 25 Crohn's disease-associated small bowel carcinoma and 25 sporadic small bowel carcinoma cases, searching for new prognostic parameters. In addition, non-tumor mucosa of celiac and Crohn's disease patients was investigated for epithelial precursor changes (hyperplastic, metaplastic or dysplastic) to help clarify carcinoma histogenesis. When compared with non-glandular structure and non-intestinal phenotype, both glandular structure and intestinal phenotype were associated with a more favorable outcome at univariable or stage- and microsatellite instability/tumor-infiltrating lymphocyte-inclusive multivariable analysis. The prognostic power of histological structure was independent of the clinical groups while the non-intestinal phenotype, associated with poor outcome, was dominant among Crohn's disease-associated carcinoma. Both nuclear β-catenin and SOX-9 were preferably expressed among celiac disease-associated carcinomas; however, they were devoid, per se, of prognostic value. We obtained findings supporting an origin of celiac disease-associated carcinoma in SOX-9-positive immature hyperplastic crypts, partly through flat β-catenin-positive dysplasia, and of Crohn's disease-associated carcinoma in a metaplastic (gastric and/or pancreatobiliary-type) mucosa, often through dysplastic polypoid growths of metaplastic phenotype. In conclusion, despite their common origin in a chronically inflamed mucosa, celiac disease-associated and Crohn's disease-associated small bowel carcinomas differ substantially in histological structure, phenotype, microsatellite instability/tumor-infiltrating lymphocyte status, Wnt pathway activation, mucosal precursor lesions and prognosis.

Small glutamine-rich tetratricopeptide repeat-containing protein alpha is present in human ovaries but may not be differentially expressed in relation to polycystic ovary syndrome.

PubMed

Butler, Miriam S; Yang, Xing; Ricciardelli, Carmela; Liang, Xiaoyan; Norman, Robert J; Tilley, Wayne D; Hickey, Theresa E

2013-06-01

To evaluate the expression and function of small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA), an androgen receptor (AR) molecular chaperone, in human ovarian tissues. Examine the effect of SGTA on AR subcellular localization in granulosa tumor cells (KGN) and SGTA expression in ovarian tissues. University-based research laboratory. Archived tissues from premenopausal women and granulosa cells from infertile women receiving assisted reproduction. None. AR subcellular localization and SGTA protein or mRNA levels. SGTA and AR proteins were expressed in the cytoplasm of KGN cells and exposure to androgen stimulated AR nuclear localization. SGTA protein knockdown increased AR nuclear localization at low (0-0.1 nmol/L) but not high (1-10 nmol/L) concentrations of androgen hormone. In ovarian tissues, SGTA was localized to the cytoplasm of granulosa cells at all stages of folliculogenesis and in thecal cells of antral follicles. SGTA protein levels were similar when comparing primordial and primary follicles within core biopsies (n = 40) from women with and without polycystic ovary syndrome (PCOS). Likewise, SGTA mRNA levels were not significantly different in granulosa cells from preovulatory follicles after hyperstimulation of women with and without PCOS. SGTA is present in human ovaries and has the potential to modulate AR signalling, but it may not be differentially expressed in PCOS. Copyright © 2013 American Society for Reproductive Medicine. All rights reserved.

Flight-induced changes in gene expression in the Glanville fritillary butterfly.

PubMed

Kvist, Jouni; Mattila, Anniina L K; Somervuo, Panu; Ahola, Virpi; Koskinen, Patrik; Paulin, Lars; Salmela, Leena; Fountain, Toby; Rastas, Pasi; Ruokolainen, Annukka; Taipale, Minna; Holm, Liisa; Auvinen, Petri; Lehtonen, Rainer; Frilander, Mikko J; Hanski, Ilkka

2015-10-01

Insect flight is one of the most energetically demanding activities in the animal kingdom, yet for many insects flight is necessary for reproduction and foraging. Moreover, dispersal by flight is essential for the viability of species living in fragmented landscapes. Here, working on the Glanville fritillary butterfly (Melitaea cinxia), we use transcriptome sequencing to investigate gene expression changes caused by 15 min of flight in two contrasting populations and the two sexes. Male butterflies and individuals from a large metapopulation had significantly higher peak flight metabolic rate (FMR) than female butterflies and those from a small inbred population. In the pooled data, FMR was significantly positively correlated with genome-wide heterozygosity, a surrogate of individual inbreeding. The flight experiment changed the expression level of 1513 genes, including genes related to major energy metabolism pathways, ribosome biogenesis and RNA processing, and stress and immune responses. Males and butterflies from the population with high FMR had higher basal expression of genes related to energy metabolism, whereas females and butterflies from the small population with low FMR had higher expression of genes related to ribosome/RNA processing and immune response. Following the flight treatment, genes related to energy metabolism were generally down-regulated, while genes related to ribosome/RNA processing and immune response were up-regulated. These results suggest that common molecular mechanisms respond to flight and can influence differences in flight metabolic capacity between populations and sexes. © 2015 John Wiley & Sons Ltd.

PinX1 Is a Potential Prognostic Factor for Non-Small-Cell Lung Cancer and Inhibits Cell Proliferation and Migration

PubMed Central

Wang, Shengguang; Zhang, Hua; Zhu, Jianquan; Li, Chenguang; Zhu, Jinfang; Shi, Bowen; Zhang, Bin

2017-01-01

PinX1 has been identified as a suppressor of telomerase enzymatic activity. However, the tumour-suppressive roles of PinX1 in different types of human cancers are unclear. PinX1 expression status and its correlation with clinicopathological features in non-small-cell lung cancer (NSCLC) have not been investigated. Accordingly, in this study, we aimed to evaluate the roles of PinX1 in NSCLC. PinX1 expression status was examined by immunohistochemistry using tissue microarray from a total of 158 patients. Correlations among PinX1 expression, clinicopathological variables, and patient survival were analysed. Furthermore, we overexpressed PinX1 in NSCLC cells and tested telomerase activity using real-time quantitative telomeric repeat amplification protocol (qTRAP) assays. Proliferation and migration of NSCLC cells were examined using the MTS method, wound healing assays, and transwell assays, respectively. Our results showed that negative PinX1 expression was associated with a poor prognosis in NSCLC. Sex, smoking status, lymph gland status, subcarinal lymph node status, pathological stage, and PinX1 expression were related to survival. PinX1 was not an independent prognostic factor in NSCLC. PinX1 overexpression inhibited proliferation and migration in NSCLC cells by suppressing telomerase activity. Our findings suggested that PinX1 could be a potential tumour suppressor in NSCLC and that loss of PinX1 promoted NSCLC progression. PMID:28815183

Expression of the Sinorhizobium meliloti small RNA gene mmgR is controlled by the nitrogen source.

PubMed

Ceizel Borella, Germán; Lagares, Antonio; Valverde, Claudio

2016-05-01

Small non-coding regulatory RNAs (sRNAs) are key players in post-transcriptional regulation of gene expression. Hundreds of sRNAs have been identified in Sinorhizobium meliloti, but their biological function remains unknown for most of them. In this study, we characterized the expression pattern of the gene encoding the 77-nt sRNA MmgR in S. meliloti strain 2011. A chromosomal transcriptional reporter fusion (PmmgR-gfp) showed that the mmgR promoter is active along different stages of the interaction with alfalfa roots. In pure cultures, PmmgR-gfp activity paralleled the sRNA abundance indicating that mmgR expression is primarily controlled at the level of transcriptional initiation. PmmgR-gfp activity was higher during growth in rhizobial defined medium (RDM) than in TY medium. Furthermore, PmmgR-gfp was induced at 60 min after shifting growing cells from TY to RDM medium, i.e. shorter than the cell doubling time. In defined RDM medium containing NO3 (-), both PmmgR-gfp and MmgR level were repressed by the addition of tryptone or single amino acids, suggesting that mmgR expression depends on the cellular nitrogen (N) status. In silico analysis failed to detect conserved motifs upstream the promoter RNA polymerase binding site, but revealed a strongly conserved motif centered at -28 that may be linked to the observed regulatory pattern by the N source. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Expression of proinflammatory cytokine interleukin-6 in tissue samples of human prostate obtained by needle biopsy.

PubMed

Miličević, Nevenka; Mrčela, Milanka; Galić, Josip; Marjanović, Ksenija

2015-11-01

Interleukin-6 (IL-6) has been associated with the development of prostate cancer. The aim of the study was to clarify whether IL-6 expression in prostate tissue could be a useful marker in differentiation of prostate diseases in small foci by pathologist visual scoring. Archival paraffin-embedded specimens of benign prostate hyperplasia (BPH), high-grade prostatic intraepithelial neoplasia (PIN), prostatitis and prostate adenocarcinoma were studied by immunohistochemistry with a mouse monoclonal antibody IL-6 using the streptavidin-biotin method. Significantly, lower IL-6 immunoreactivity was observed in normal epithelial cells (p=0.000) and basal cells (p=0.000) in the samples of prostate adenocarcinoma in comparison to the samples with BPH, PIN and prostatitis. There was no significant difference in IL-6 expression in malignant and premalignant cells (p=0.814) as well as in stromal cells among the four diagnoses (p=0.22). IL-6 was expressed in normal epithelial cells, premalignant epithelial cells and malignant epithelial cells as well as in stromal cells. However, in our research IL-6 was of limited utility as a single marker for differential diagnosis of the prostate diseases in small foci needle biopsy by pathologist visual scoring. The standardization of immunohistochemical (IHC) staining protocol for IL-6 is required to determine IL-6 expression in order to avoid possible misinterpretation of the IHC results. Copyright © 2015 Elsevier GmbH. All rights reserved.

Implicit theories about interrelations of anger components in 25 countries.

PubMed

Alonso-Arbiol, Itziar; van de Vijver, Fons J R; Fernandez, Itziar; Paez, Dario; Campos, Miryam; Carrera, Pilar

2011-02-01

We were interested in the cross-cultural comparison of implicit theories of the interrelations of eight anger components (antecedents, body sensations, cognitive reactions, verbal expressions, nonverbal expressions, interpersonal responses, and primary and secondary self-control). Self-report scales of each of these components were administered to a total of 5,006 college students in 25 countries. Equivalence of the scales was supported in that scales showed acceptable congruence coefficients in almost all comparisons. A multigroup confirmatory factor model with three latent variables (labeled internal processes, behavioral outcomes, and self-control mechanisms) could well account for the interrelations of the eight observed variables; measurement and structural weights were invariant. Behavioral outcomes and self-control mechanisms were only associated through their common dependence on internal processes. Verbal expressions and cognitive reactions showed the largest cross-cultural differences in means, whereas self-control mechanisms scales showed the smallest differences. Yet, cultural differences between the countries were small. It is concluded that anger, as measured by these scales, shows more pronounced cross-cultural similarities than differences in terms of both interrelations and mean score levels. PsycINFO Database Record (c) 2011 APA, all rights reserved.

Emotional Expressiveness during Peer Conflicts: A Predictor of Social Maladjustment among High-Risk Preschoolers.

ERIC Educational Resources Information Center

Miller, Alison L.; Olson, Sheryl L.

This study examined the relationship of individual differences in peer sociometric status and teacher ratings of disruptive behavior, and preschool boys' emotion displays during conflicts with mixed-sex peers. Sixty 4- and 5-year-old boys from low-income families were videotaped with a small group of classmates in a Head Start preschool classroom.…

Post-transcriptional bursting in genes regulated by small RNA molecules

NASA Astrophysics Data System (ADS)

Rodrigo, Guillermo

2018-03-01

Gene expression programs in living cells are highly dynamic due to spatiotemporal molecular signaling and inherent biochemical stochasticity. Here we study a mechanism based on molecule-to-molecule variability at the RNA level for the generation of bursts of protein production, which can lead to heterogeneity in a cell population. We develop a mathematical framework to show numerically and analytically that genes regulated post transcriptionally by small RNA molecules can exhibit such bursts due to different states of translation activity (on or off), mostly revealed in a regime of few molecules. We exploit this framework to compare transcriptional and post-transcriptional bursting and also to illustrate how to tune the resulting protein distribution with additional post-transcriptional regulations. Moreover, because RNA-RNA interactions are predictable with an energy model, we define the kinetic constants of on-off switching as functions of the two characteristic free-energy differences of the system, activation and formation, with a nonequilibrium scheme. Overall, post-transcriptional bursting represents a distinctive principle linking gene regulation to gene expression noise, which highlights the importance of the RNA layer beyond the simple information transfer paradigm and significantly contributes to the understanding of the intracellular processes from a first-principles perspective.

Production of recombinant antigens and antibodies in Nicotiana benthamiana using 'magnifection' technology: GMP-compliant facilities for small- and large-scale manufacturing.

PubMed

Klimyuk, Victor; Pogue, Gregory; Herz, Stefan; Butler, John; Haydon, Hugh

2014-01-01

This review describes the adaptation of the plant virus-based transient expression system, magnICON(®) for the at-scale manufacturing of pharmaceutical proteins. The system utilizes so-called "deconstructed" viral vectors that rely on Agrobacterium-mediated systemic delivery into the plant cells for recombinant protein production. The system is also suitable for production of hetero-oligomeric proteins like immunoglobulins. By taking advantage of well established R&D tools for optimizing the expression of protein of interest using this system, product concepts can reach the manufacturing stage in highly competitive time periods. At the manufacturing stage, the system offers many remarkable features including rapid production cycles, high product yield, virtually unlimited scale-up potential, and flexibility for different manufacturing schemes. The magnICON system has been successfully adaptated to very different logistical manufacturing formats: (1) speedy production of multiple small batches of individualized pharmaceuticals proteins (e.g. antigens comprising individualized vaccines to treat NonHodgkin's Lymphoma patients) and (2) large-scale production of other pharmaceutical proteins such as therapeutic antibodies. General descriptions of the prototype GMP-compliant manufacturing processes and facilities for the product formats that are in preclinical and clinical testing are provided.

Small RNA-mediated responses to low- and high-temperature stresses in cotton

PubMed Central

Wang, Qiongshan; Liu, Nian; Yang, Xiyan; Tu, Lili; Zhang, Xianlong

2016-01-01

MicroRNAs (miRNAs) are one class of endogenous non-coding RNAs modulating the expression of target genes involved in plant development and stress tolerance, by degrading mRNA or repressing translation. In this study, small RNA and mRNA degradome sequencing were used to identify low- and high-temperature stress-responsive miRNAs and their targets in cotton (Gossypium hirsutum). Cotton seedlings were treated under different temperature conditions (4, 12, 25, 35, and 42 °C) and then the effects were investigated. In total, 319 known miRNAs and 800 novel miRNAs were identified, and 168 miRNAs were differentially expressed between different treatments. The targets of these miRNAs were further analysed by degradome sequencing. Based on studies from Gene Ontology and Kyoto Encyclopedia of Genes and Genomes, the majority of the miRNAs are from genes that are likely involved in response to hormone stimulus, oxidation-reduction reaction, photosynthesis, plant–pathogen interaction and plant hormone signal transduction pathways. This study provides new insight into the molecular mechanisms of plant response to extreme temperature stresses, and especially the roles of miRNAs under extreme temperatures. PMID:27752116

RNA-seq reveals distinctive RNA profiles of small extracellular vesicles from different human liver cancer cell lines

PubMed Central

Berardocco, Martina; Radeghieri, Annalisa; Busatto, Sara; Gallorini, Marialucia; Raggi, Chiara; Gissi, Clarissa; D’Agnano, Igea; Bergese, Paolo; Felsani, Armando; Berardi, Anna C.

2017-01-01

Liver cancer (LC) is one of the most common cancers and represents the third highest cause of cancer-related deaths worldwide. Extracellular vesicle (EVs) cargoes, which are selectively enriched in RNA, offer great promise for the diagnosis, prognosis and treatment of LC. Our study analyzed the RNA cargoes of EVs derived from 4 liver-cancer cell lines: HuH7, Hep3B, HepG2 (hepato-cellular carcinoma) and HuH6 (hepatoblastoma), generating two different sets of sequencing libraries for each. One library was size-selected for small RNAs and the other targeted the whole transcriptome. Here are reported genome wide data of the expression level of coding and non-coding transcripts, microRNAs, isomiRs and snoRNAs providing the first comprehensive overview of the extracellular-vesicle RNA cargo released from LC cell lines. The EV-RNA expression profiles of the four liver cancer cell lines share a similar background, but cell-specific features clearly emerge showing the marked heterogeneity of the EV-cargo among the individual cell lines, evident both for the coding and non-coding RNA species. PMID:29137313

Significance of aquaporins’ expression in the prognosis of gastric cancer

PubMed Central

Thapa, Saroj; Chetry, Mandika; Huang, Kaiyu; Peng, Yangpei; Wang, Jinsheng; Wang, Jiaoni; Zhou, Yingying; Shen, Yigen; Xue, Yangjing; Ji, Kangting

2018-01-01

Gastric carcinoma is one of the most lethal malignancy at present with leading cause of cancer-related deaths worldwide. Aquaporins (AQPs) are a family of small, integral membrane proteins, which have been evidenced to play a crucial role in cell migration and proliferation of different cancer cells including gastric cancers. However, the aberrant expression of specific AQPs and its correlation to detect predictive and prognostic significance in gastric cancer remains elusive. In the present study, we comprehensively explored immunohistochemistry based map of protein expression profiles in normal tissues, cancer and cell lines from publicly available Human Protein Atlas (HPA) database. Moreover, to improve our understanding of general gastric biology and guide to find novel predictive prognostic gastric cancer biomarker, we also retrieved ‘The Kaplan–Meier plotter’ (KM plotter) online database with specific AQPs mRNA to overall survival (OS) in different clinicopathological features. We revealed that ubiquitous expression of AQPs protein can be effective tools to generate gastric cancer biomarker. Furthermore, high level AQP3, AQP9, and AQP11 mRNA expression were correlated with better OS in all gastric patients, whereas AQP0, AQP1, AQP4, AQP5, AQP6, AQP8, and AQP10 mRNA expression were associated with poor OS. With regard to the clinicopathological features including Laurens classification, clinical stage, human epidermal growth factor receptor 2 (HER2) status, and different treatment strategy, we could illustrate significant role of individual AQP mRNA expression in the prognosis of gastric cancer patients. Thus, our results indicated that AQP’s protein and mRNA expression in gastric cancer patients provide effective role to predict prognosis and act as an essential agent to therapeutic strategy. PMID:29678898

Expression of anti-Mullerian hormone in hens selected for different ovulation rates.

PubMed

Johnson, P A; Kent, T R; Urick, M E; Trevino, L S; Giles, J R

2009-05-01

In hens, the granulosa layer is the primary source of anti-Mullerian hormone (AMH), as it is in mammals. Small follicles express the greatest amount of Amh mRNA with less in the larger follicles. Laying hens have a distinct ovarian hierarchy of follicles while broiler breeder hens often have excessive follicle growth with a disrupted hierarchy. The objective of Experiment 1 was to examine Amh expression in two strains of hens differing in ovulatory efficiency. Amh expression was greater (P<0.01) in broiler breeder hens (n=6) as compared with laying hens (n=6). Experiment 2 was designed to examine whether alterations in follicular development due to diet, within the broiler breeder hens, were correlated with changes in the expression of Amh. Restricted feeding (RF) in broiler breeder hens promotes optimal follicular development. Egg production in broiler breeder hens on full feed (FF; n=8) was 78% that of hens on RF (n=9). The number of large follicles (P<0.05), total ovarian weight (P<0.01), and Amh mRNA expression were greater in FF hens as compared with RF hens (P<0.01). There was no difference in FSH receptor expression between the two groups. A direct nutritional effect was not supported because culture of granulosa cells with varying concentrations of glucose and insulin showed no effect on granulosa Amh expression. Finally, testis-conditioned medium resulted in a dose-related increase in granulosa cell proliferation, which could be inhibited by preincubation with AMH antibody. AMH may enhance granulosa cell proliferation through an autocrine or paracrine mechanism although excessive AMH may inhibit optimal follicle selection.

Argonautes promote male fertility and provide a paternal memory of germline gene expression in C. elegans

PubMed Central

Conine, Colin C.; Moresco, James J.; Gu, Weifeng; Shirayama, Masaki; Conte, Darryl; Yates, John R.; Mello, Craig C.

2014-01-01

SUMMARY During each life cycle germ cells preserve and pass on both genetic and epigenetic information. In C. elegans, the ALG-3/4 Argonaute proteins are expressed during male gametogenesis and promote male fertility. Here we show that the CSR-1 Argonaute functions with ALG-3/4 to positively regulate target genes required for spermiogenesis. Our findings suggest that ALG-3/4 functions during spermatogenesis to amplify a small-RNA signal that represents an epigenetic memory of male-specific gene expression. CSR-1, which is abundant in mature sperm, appears to transmit this memory to offspring. Surprisingly, in addition to small RNAs targeting male-specific genes, we show that males also harbor an extensive repertoire of CSR-1 small RNAs targeting oogenesis-specific mRNAs. Together these findings suggest that C. elegans sperm transmit not only the genome but also epigenetic binary signals in the form of Argonaute/small-RNA complexes that constitute a memory of gene expression in preceding generations. PMID:24360276

The cationic small molecule GW4869 is cytotoxic to high phosphatidylserine-expressing myeloma cells.

PubMed

Vuckovic, Slavica; Vandyke, Kate; Rickards, David A; McCauley Winter, Padraig; Brown, Simon H J; Mitchell, Todd W; Liu, Jun; Lu, Jun; Askenase, Philip W; Yuriev, Elizabeth; Capuano, Ben; Ramsland, Paul A; Hill, Geoffrey R; Zannettino, Andrew C W; Hutchinson, Andrew T

2017-05-01

We have discovered that a small cationic molecule, GW4869, is cytotoxic to a subset of myeloma cell lines and primary myeloma plasma cells. Biochemical analysis revealed that GW4869 binds to anionic phospholipids such as phosphatidylserine - a lipid normally confined to the intracellular side of the cell membrane. However, interestingly, phosphatidylserine was expressed on the surface of all myeloma cell lines tested (n = 12) and 9/15 primary myeloma samples. Notably, the level of phosphatidylserine expression correlated well with sensitivity to GW4869. Inhibition of cell surface phosphatidylserine exposure with brefeldin A resulted in resistance to GW4869. Finally, GW4869 was shown to delay the growth of phosphatidylserine-high myeloma cells in vivo. To the best of our knowledge, this is the first example of using a small molecule to target phosphatidylserine on malignant cells. This study may provide the rationale for the development of phosphatidylserine-targeting small molecules for the treatment of surface phosphatidylserine-expressing cancers. © 2017 John Wiley & Sons Ltd.

A complex dominance hierarchy is controlled by polymorphism of small RNAs and their targets.

PubMed

Yasuda, Shinsuke; Wada, Yuko; Kakizaki, Tomohiro; Tarutani, Yoshiaki; Miura-Uno, Eiko; Murase, Kohji; Fujii, Sota; Hioki, Tomoya; Shimoda, Taiki; Takada, Yoshinobu; Shiba, Hiroshi; Takasaki-Yasuda, Takeshi; Suzuki, Go; Watanabe, Masao; Takayama, Seiji

2016-12-22

In diploid organisms, phenotypic traits are often biased by effects known as Mendelian dominant-recessive interactions between inherited alleles. Phenotypic expression of SP11 alleles, which encodes the male determinants of self-incompatibility in Brassica rapa, is governed by a complex dominance hierarchy 1-3 . Here, we show that a single polymorphic 24 nucleotide small RNA, named SP11 methylation inducer 2 (Smi2), controls the linear dominance hierarchy of the four SP11 alleles (S 44 > S 60 > S 40 > S 29 ). In all dominant-recessive interactions, small RNA variants derived from the linked region of dominant SP11 alleles exhibited high sequence similarity to the promoter regions of recessive SP11 alleles and acted in trans to epigenetically silence their expression. Together with our previous study 4 , we propose a new model: sequence similarity between polymorphic small RNAs and their target regulates mono-allelic gene expression, which explains the entire five-phased linear dominance hierarchy of the SP11 phenotypic expression in Brassica.

Brief Report: A mass spectrometry assay to simultaneously analyze ROS1 and RET fusion gene expression in non-small cell lung cancer

PubMed Central

Wijesinghe, Priyanga; Bepler, Gerold

2014-01-01

Introduction ROS1 and RET gene fusions were recently discovered in non-small cell lung cancer (NSCLC) as potential therapeutic targets with small molecule kinase inhibitors. The conventional screening methods of these fusions are time consuming and require samples of high quality and quantity. Here, we describe a novel and efficient method by coupling the power of multiplexing PCR and the sensitivity of mass spectrometry. Methods The multiplex mass spectrometry platform simultaneously tests samples for the expression of nine ROS1 and six RET fusion genes. The assay incorporates detection of wild-type exon junctions immediately upstream and downstream of the fusion junction to exclude false negative results. To flag false positives, the system also comprises two independent assays for each fusion gene junction. Results The characteristic mass spectrometric peaks of the gene fusions were obtained using engineered plasmid constructs. Specific assays targeting the wild-type gene exon junctions were validated using cDNA from lung tissue of healthy individuals. The system was further validated using cDNA derived from NSCLC cell lines that express endogenous fusion genes. The expressed ROS1-SLC34A2 and CCDC6-RET gene fusions from the NSCLC cell lines HCC78 and LC-2/ad, respectively, were accurately detected by the novel assay. The assay is extremely sensitive, capable of detecting an event in test specimens containing 0.5% positive tumors. Conclusion The novel multiplexed assay is robustly capable of detecting 15 different clinically relevant RET and ROS1 fusion variants. The benefits of this detection method include exceptionally low sample input, high cost efficiency, flexibility, and rapid turnover. PMID:25384172

Evaluation of role of Notch3 signaling pathway in human lung cancer cells.

PubMed

Hassan, Wael Abdo; Yoshida, Ryoji; Kudoh, Shinji; Motooka, Yamato; Ito, Takaaki

2016-05-01

There is still a debate on the extent to which Notch3 signaling is involved in lung carcinogenesis and whether such function is dependent on cancer type or not. To evaluate Notch3 expression in different types of human lung cancer cells. Notch3 was detected in human lung cancer cell lines and in tissues. Then, small interfering RNA (siRNA) was used to down-regulate the expression of Notch3 in H69AR small cell lung carcinoma (SCLC) cells; two non-small cell lung carcinoma (NSCLC) cells; A549 adenocarcinoma (ADC); and H2170 squamous cell carcinoma (SCC). In addition, Notch3 intracellular domain (N3ICD) plasmid was transfected into H1688 human SCLC cells. We observed the effect of deregulating Notch3 signaling on the following cell properties: Notch-related proteins, cell morphology, adhesion, epithelial-mesenchymal transition (EMT), motility, proliferation and neuroendocrine (NE) features of SCLC. Notch3 is mainly expressed in NSCLC, and the expression of Notch1, Hes1 and Jagged1 is affected by Notch3. Notch3 has opposite functions in SCLC and NSCLC, being a tumor suppressor in the former and tumor promoting in the latter, in the context of cell adhesion, EMT and motility. Regarding cell proliferation, we found that inhibiting Notch3 in NSCLC decreases cell proliferation and induces apoptosis in NSCLC. Notch3 has no effect on cell proliferation or NE features of SCLC. Notch3 signaling in lung carcinoma is dependent on cell type. In SCLC, Notch3 behaves as a tumor suppressor pathway, while in NSCLC it acts as a tumor-promoting pathway.

Erythritol reduces small intestinal glucose absorption, increases muscle glucose uptake, improves glucose metabolic enzymes activities and increases expression of Glut-4 and IRS-1 in type 2 diabetic rats.

PubMed

Chukwuma, Chika Ifeanyi; Mopuri, Ramgopal; Nagiah, Savania; Chuturgoon, Anil Amichund; Islam, Md Shahidul

2017-08-02

Studies have reported that erythritol, a low or non-glycemic sugar alcohol possesses anti-hyperglycemic and anti-diabetic potentials but the underlying mode of actions is not clear. This study investigated the underlying mode of actions behind the anti-hyperglycemic and anti-diabetic potentials of erythritol using different experimental models (experiment 1, 2 and 3). Experiment 1 examined the effects of increasing concentrations (2.5-20%) of erythritol on glucose absorption and uptake in isolated rat jejunum and psoas muscle, respectively. Experiments 2 and 3 examined the effects of a single oral dose of erythritol (1 g/kg bw) on intestinal glucose absorption, gastric emptying and postprandial blood glucose increase, glucose tolerance, serum insulin level, muscle/liver hexokinase and liver glucose-6 phosphatase activities, liver and muscle glycogen contents and mRNA and protein expression of muscle Glut-4 and IRS-1 in normal and type 2 diabetic animals. Experiment 1 revealed that erythritol dose dependently enhanced muscle glucose ex vivo. Experiment 2 demonstrated that erythritol feeding delayed gastric emptying and reduced small intestinal glucose absorption as well as postprandial blood glucose rise, especially in diabetic animals. Experiment 3 showed that erythritol feeding improved glucose tolerance, muscle/liver hexokinase and liver glucose-6 phosphatase activities, glycogen storage and also modulated expression of muscle Glut-4 and IRS-1 in diabetic animals. Data suggest that erythritol may exert anti-hyperglycemic effects not only via reducing small intestinal glucose absorption, but also by increasing muscle glucose uptake, improving glucose metabolic enzymes activity and modulating muscle Glut-4 and IRS-1 mRNA and protein expression. Hence, erythritol may be a useful dietary supplement for managing hyperglycemia, particularly for T2D.

Characterization of the Small RNA Transcriptome of the Marine Coccolithophorid, Emiliania huxleyi.

PubMed

Zhang, Xiaoyu; Gamarra, Jaime; Castro, Steven; Carrasco, Estela; Hernandez, Aaron; Mock, Thomas; Hadaegh, Ahmad R; Read, Betsy A

2016-01-01

Small RNAs (smRNAs) control a variety of cellular processes by silencing target genes at the transcriptional or post-transcription level. While extensively studied in plants, relatively little is known about smRNAs and their targets in marine phytoplankton, such as Emiliania huxleyi (E. huxleyi). Deep sequencing was performed of smRNAs extracted at different time points as E. huxleyi cells transition from logarithmic to stationary phase growth in batch culture. Computational analyses predicted 18 E. huxleyi specific miRNAs. The 18 miRNA candidates and their precursors vary in length (18-24 nt and 71-252 nt, respectively), genome copy number (3-1,459), and the number of genes targeted (2-107). Stem-loop real time reverse transcriptase (RT) PCR was used to validate miRNA expression which varied by nearly three orders of magnitude when growth slows and cells enter stationary phase. Stem-loop RT PCR was also used to examine the expression profiles of miRNA in calcifying and non-calcifying cultures, and a small subset was found to be differentially expressed when nutrients become limiting and calcification is enhanced. In addition to miRNAs, endogenous small RNAs such as ra-siRNAs, ta-siRNAs, nat-siRNAs, and piwiRNAs were predicted along with the machinery for the biogenesis and processing of si-RNAs. This study is the first genome-wide investigation smRNAs pathways in E. huxleyi. Results provide new insights into the importance of smRNAs in regulating aspects of physiological growth and adaptation in marine phytoplankton and further challenge the notion that smRNAs evolved with multicellularity, expanding our perspective of these ancient regulatory pathways.

Characterization of the Small RNA Transcriptome of the Marine Coccolithophorid, Emiliania huxleyi

PubMed Central

Zhang, Xiaoyu; Gamarra, Jaime; Castro, Steven; Carrasco, Estela; Hernandez, Aaron; Mock, Thomas; Hadaegh, Ahmad R.; Read, Betsy A.

2016-01-01

Small RNAs (smRNAs) control a variety of cellular processes by silencing target genes at the transcriptional or post-transcription level. While extensively studied in plants, relatively little is known about smRNAs and their targets in marine phytoplankton, such as Emiliania huxleyi (E. huxleyi). Deep sequencing was performed of smRNAs extracted at different time points as E. huxleyi cells transition from logarithmic to stationary phase growth in batch culture. Computational analyses predicted 18 E. huxleyi specific miRNAs. The 18 miRNA candidates and their precursors vary in length (18–24 nt and 71–252 nt, respectively), genome copy number (3–1,459), and the number of genes targeted (2–107). Stem-loop real time reverse transcriptase (RT) PCR was used to validate miRNA expression which varied by nearly three orders of magnitude when growth slows and cells enter stationary phase. Stem-loop RT PCR was also used to examine the expression profiles of miRNA in calcifying and non-calcifying cultures, and a small subset was found to be differentially expressed when nutrients become limiting and calcification is enhanced. In addition to miRNAs, endogenous small RNAs such as ra-siRNAs, ta-siRNAs, nat-siRNAs, and piwiRNAs were predicted along with the machinery for the biogenesis and processing of si-RNAs. This study is the first genome-wide investigation smRNAs pathways in E. huxleyi. Results provide new insights into the importance of smRNAs in regulating aspects of physiological growth and adaptation in marine phytoplankton and further challenge the notion that smRNAs evolved with multicellularity, expanding our perspective of these ancient regulatory pathways. PMID:27101007

Expanding the action of duplex RNAs into the nucleus: redirecting alternative splicing

PubMed Central

Liu, Jing; Hu, Jiaxin; Corey, David R.

2012-01-01

Double-stranded RNAs are powerful agents for silencing gene expression in the cytoplasm of mammalian cells. The potential for duplex RNAs to control expression in the nucleus has received less attention. Here, we investigate the ability of small RNAs to redirect splicing. We identify RNAs targeting an aberrant splice site that restore splicing and production of functional protein. RNAs can target sequences within exons or introns and affect the inclusion of exons within SMN2 and dystrophin, genes responsible for spinal muscular atrophy and Duchenne muscular dystrophy, respectively. Duplex RNAs recruit argonaute 2 (AGO2) to pre-mRNA transcripts and altered splicing requires AGO2 expression. AGO2 promotes transcript cleavage in the cytoplasm, but recruitment of AGO2 to pre-mRNAs does not reduce transcript levels, exposing a difference between cytoplasmic and nuclear pathways. Involvement of AGO2 in splicing, a classical nuclear process, reinforces the conclusion from studies of RNA-mediated transcriptional silencing that RNAi pathways can be adapted to function in the mammalian nucleus. These data provide a new strategy for controlling splicing and expand the reach of small RNAs within the nucleus of mammalian cells. PMID:21948593

Genome-wide discovery and differential regulation of conserved and novel microRNAs in chickpea via deep sequencing.

PubMed

Jain, Mukesh; Chevala, V V S Narayana; Garg, Rohini

2014-11-01

MicroRNAs (miRNAs) are essential components of complex gene regulatory networks that orchestrate plant development. Although several genomic resources have been developed for the legume crop chickpea, miRNAs have not been discovered until now. For genome-wide discovery of miRNAs in chickpea (Cicer arietinum), we sequenced the small RNA content from seven major tissues/organs employing Illumina technology. About 154 million reads were generated, which represented more than 20 million distinct small RNA sequences. We identified a total of 440 conserved miRNAs in chickpea based on sequence similarity with known miRNAs in other plants. In addition, 178 novel miRNAs were identified using a miRDeep pipeline with plant-specific scoring. Some of the conserved and novel miRNAs with significant sequence similarity were grouped into families. The chickpea miRNAs targeted a wide range of mRNAs involved in diverse cellular processes, including transcriptional regulation (transcription factors), protein modification and turnover, signal transduction, and metabolism. Our analysis revealed several miRNAs with differential spatial expression. Many of the chickpea miRNAs were expressed in a tissue-specific manner. The conserved and differential expression of members of the same miRNA family in different tissues was also observed. Some of the same family members were predicted to target different chickpea mRNAs, which suggested the specificity and complexity of miRNA-mediated developmental regulation. This study, for the first time, reveals a comprehensive set of conserved and novel miRNAs along with their expression patterns and putative targets in chickpea, and provides a framework for understanding regulation of developmental processes in legumes. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Molecular Control of TiO2-NPs Toxicity Formation at Predicted Environmental Relevant Concentrations by Mn-SODs Proteins

PubMed Central

Wu, Qiuli; Li, Yiping; Tang, Meng; Ye, Boping; Wang, Dayong

2012-01-01

With growing concerns of the safety of nanotechnology, the in vivo toxicity of nanoparticles (NPs) at environmental relevant concentrations has drawn increasing attentions. We investigated the possible molecular mechanisms of titanium nanoparticles (Ti-NPs) in the induction of toxicity at predicted environmental relevant concentrations. In nematodes, small sizes (4 nm and 10 nm) of TiO2-NPs induced more severe toxicities than large sizes (60 nm and 90 nm) of TiO2-NPs on animals using lethality, growth, reproduction, locomotion behavior, intestinal autofluorescence, and reactive oxygen species (ROS) production as endpoints. Locomotion behaviors could be significantly decreased by exposure to 4-nm and 10-nm TiO2-NPs at concentration of 1 ng/L in nematodes. Among genes required for the control of oxidative stress, only the expression patterns of sod-2 and sod-3 genes encoding Mn-SODs in animals exposed to small sizes of TiO2-NPs were significantly different from those in animals exposed to large sizes of TiO2-NPs. sod-2 and sod-3 gene expressions were closely correlated with lethality, growth, reproduction, locomotion behavior, intestinal autofluorescence, and ROS production in TiO2-NPs-exposed animals. Ectopically expression of human and nematode Mn-SODs genes effectively prevented the induction of ROS production and the development of toxicity of TiO2-NPs. Therefore, the altered expression patterns of Mn-SODs may explain the toxicity formation for different sizes of TiO2-NPs at predicted environmental relevant concentrations. In addition, we demonstrated here a strategy to investigate the toxicological effects of exposure to NPs upon humans by generating transgenic strains in nematodes for specific human genes. PMID:22973466

A small population of resident limb bud mesenchymal cells express few MSC-associated markers, but the expression of these markers is increased immediately after cell culture.

PubMed

Marín-Llera, Jessica Cristina; Chimal-Monroy, Jesús

2018-05-01

Skeletal progenitors are derived from resident limb bud mesenchymal cells of the vertebrate embryos. However, it remains poorly understood if they represent stem cells, progenitors, or multipotent mesenchymal stromal cells (MSC). Derived-MSC of different adult tissues under in vitro experimental conditions can differentiate into the same cellular lineages that are present in the limb. Here, comparing non-cultured versus cultured mesenchymal limb bud cells, we determined the expression of MSC-associated markers, the in vitro differentiation capacity and their gene expression profile. Results showed that in freshly isolated limb bud mesenchymal cells, the proportion of cells expressing Sca1, CD44, CD105, CD90, and CD73 is very low and a low expression of lineage-specific genes was observed. However, recently seeded limb bud mesenchymal cells acquired Sca1 and CD44 markers and the expression of the key differentiation genes Runx2 and Sox9, while Scx and Pparg genes decreased. Also, their chondrogenic differentiation capacity decreased through cellular passages while the osteogenic increased. Our findings suggest that the modification of the cell adhesion process through the in vitro method changed the limb mesenchymal cell immunophenotype leading to the expression and maintenance of common MSC-associated markers. These findings could have a significant impact on MSC study and isolation strategy because they could explain common variations observed in the MSC immunophenotype in different tissues. © 2018 International Federation for Cell Biology.

Inflammation and nerve injury induce expression of pancreatitis-associated protein-II in primary sensory neurons.

PubMed

He, Shao-Qiu; Yao, Jun-Ru; Zhang, Fang-Xiong; Wang, Qiong; Bao, Lan; Zhang, Xu

2010-04-26

Pancreatitis-associated protein (PAP)-I and -II, lectin-related secretory proteins, are members of the regenerating gene (Reg) family. Although expression of PAP-I was found in the dorsal root ganglion (DRG) neurons following peripheral nerve injury and cystitis, whether PAP-II could be expressed in DRG neurons in chronic pain models remains unclear. The present study shows an inflammation- and nerve injury-triggered expression of PAP-II in rat DRG neurons. In situ hybridization showed that only a few DRG neurons normally contained PAP-I and -II mRNAs. After peripheral inflammation, PAP-I and -II mRNAs were present in over half of small DRG neurons. Such an elevated expression of PAP-I and -II reached the peak level on the second day. Immunostaining showed that the expression of PAP-II was mostly increased in the isolectin B4-positive subset of small DRG neurons after inflammation. Furthermore, the expression of PAP-II was also induced in DRG neurons after peripheral nerve injury. Interestingly, PAP-II expression was shifted from small neurons on day 2 to large DRG neurons that expressed neuropeptide Y during the later post-injury days. These results suggest that PAP-II may play potential roles in the modulation of spinal sensory pathways in pathological pain states.

Bet-hedging in bacteriocin producing Escherichia coli populations: the single cell perspective

NASA Astrophysics Data System (ADS)

Bayramoglu, Bihter; Toubiana, David; van Vliet, Simon; Inglis, R. Fredrik; Shnerb, Nadav; Gillor, Osnat

2017-02-01

Production of public goods in biological systems is often a collaborative effort that may be detrimental to the producers. It is therefore sustainable only if a small fraction of the population shoulders the cost while the majority reap the benefits. We modelled this scenario using Escherichia coli populations producing colicins, an antibiotic that kills producer cells’ close relatives. Colicin expression is a costly trait, and it has been proposed that only a small fraction of the population actively expresses the antibiotic. Colicinogenic populations were followed at the single-cell level using time-lapse microscopy, and showed two distinct, albeit dynamic, subpopulations: the majority silenced colicin expression, while a small fraction of elongated, slow-growing cells formed colicin-expressing hotspots, placing a significant burden on expressers. Moreover, monitoring lineages of individual colicinogenic cells showed stochastic switching between expressers and non-expressers. Hence, colicin expressers may be engaged in risk-reducing strategies—or bet-hedging—as they balance the cost of colicin production with the need to repel competitors. To test the bet-hedging strategy in colicin-mediated interactions, competitions between colicin-sensitive and producer cells were simulated using a numerical model, demonstrating a finely balanced expression range that is essential to sustaining the colicinogenic population.

Prevalence of Enhancer of Zeste Homolog 2 in Patients with Resected Small Cell Lung Cancer.

PubMed

Toyokawa, Gouji; Takada, Kazuki; Tagawa, Tetsuzo; Kinoshita, Fumihiko; Kozuma, Yuka; Matsubara, Taichi; Haratake, Naoki; Takamori, Shinkichi; Akamine, Takaki; Hirai, Fumihiko; Yamada, Yuichi; Hamamoto, Ryuji; Oda, Yoshinao; Maehara, Yoshihiko

2018-06-01

Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that is deeply involved in cancer pathogenesis. Although clinicopathological significance of EZH2 in non-small cell lung cancer has been gradually elucidated, such significance in small cell lung cancer (SCLC) has yet to be fully investigated. Forty patients with resected SCLC were analyzed for EZH2. EZH2 expression was evaluated using the Allred score (0-8) and was classified into negative (0-6) and positive (7 and 8). We evaluated the association between EZH2 and the clinicopathological characteristics and postoperative survivals. Among 40 patients, 15 (37.5%) and 25 (62.5%) were classified as being negative and positive for EZH2, respectively. Fisher's exact test demonstrated no significant associations between the positivity for EZH2 and clinicopathological characteristics. No significant differences were observed in recurrence-free and overall survivals between EZH2-negative/low and EZH2-high patients. EZH2 was frequently observed in patients with resected SCLC, but no significant associations were found between its expression and the clinicopathological characteristics and postoperative survivals. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation.

PubMed

Lanik, Wyatt E; Xu, Lily; Luke, Cliff J; Hu, Elise Z; Agrawal, Pranjal; Liu, Victoria S; Kumar, Rajesh; Bolock, Alexa M; Ma, Congrong; Good, Misty

2018-02-15

Human small intestinal enteroids are derived from the crypts and when grown in a stem cell niche contain all of the epithelial cell types. The ability to establish human enteroid ex vivo culture systems are important to model intestinal pathophysiology and to study the particular cellular responses involved. In recent years, enteroids from mice and humans are being cultured, passaged, and banked away for future use in several laboratories across the world. This enteroid platform can be used to test the effects of various treatments and drugs and what effects are exerted on different cell types in the intestine. Here, a protocol for establishing primary stem cell-derived small intestinal enteroids derived from neonatal mice and premature human intestine is provided. Moreover, this enteroid culture system was utilized to test the effects of species-specific breast milk. Mouse breast milk can be obtained efficiently using a modified human breast pump and expressed mouse milk can then be used for further research experiments. We now demonstrate the effects of expressed mouse, human, and donor breast milk on the growth and proliferation of enteroids derived from neonatal mice or premature human small intestine.

Small RNA Transcriptome of Hibiscus Syriacus Provides Insights into the Potential Influence of microRNAs in Flower Development and Terpene Synthesis.

PubMed

Kim, Taewook; Park, June Hyun; Lee, Sang-Gil; Kim, Soyoung; Kim, Jihyun; Lee, Jungho; Shin, Chanseok

2017-08-01

MicroRNAs (miRNAs) are essential small RNA molecules that regulate the expression of target mRNAs in plants and animals. Here, we aimed to identify miRNAs and their putative targets in Hibiscus syriacus , the national flower of South Korea. We employed high-throughput sequencing of small RNAs obtained from four different tissues ( i.e. , leaf, root, flower, and ovary) and identified 33 conserved and 30 novel miRNA families, many of which showed differential tissue-specific expressions. In addition, we computationally predicted novel targets of miRNAs and validated some of them using 5' rapid amplification of cDNA ends analysis. One of the validated novel targets of miR477 was a terpene synthase, the primary gene involved in the formation of disease-resistant terpene metabolites such as sterols and phytoalexins. In addition, a predicted target of conserved miRNAs, miR396, is SHORT VEGETATIVE PHASE , which is involved in flower initiation and is duplicated in H. syriacus . Collectively, this study provides the first reliable draft of the H. syriacus miRNA transcriptome that should constitute a basis for understanding the biological roles of miRNAs in H. syriacus.

RsmV a small non-coding regulatory RNA in Pseudomonas aeruginosa that sequesters RsmA and RsmF from target mRNAs.

PubMed

Janssen, Kayley H; Diaz, Manisha R; Gode, Cindy J; Wolfgang, Matthew C; Yahr, Timothy L

2018-06-04

The Gram-negative opportunistic pathogen Pseudomonas aeruginosa has distinct genetic programs that favor either acute or chronic virulence gene expression. Acute virulence is associated with twitching and swimming motility, expression of a type III secretion system (T3SS), and the absence of alginate, Psl, or Pel polysaccharide production. Traits associated with chronic infection include growth as a biofilm, reduced motility, and expression of a type VI secretion system (T6SS). The Rsm post-transcriptional regulatory system plays important roles in the inverse control of phenotypes associated with acute and chronic virulence. RsmA and RsmF are RNA-binding proteins that interact with target mRNAs to control gene expression at the post-transcriptional level. Previous work found that RsmA activity is controlled by at least three small, non-coding regulatory RNAs (RsmW, RsmY, and RsmZ). In this study, we took an in-silico approach to identify additional sRNAs that might function in the sequestration of RsmA and/or RsmF and identified RsmV, a 192 nt transcript with four predicted RsmA/RsmF consensus binding sites. RsmV is capable of sequestering RsmA and RsmF in vivo to activate translation of tssA1 , a component of the T6SS, and to inhibit T3SS gene expression. Each of the predicted RsmA/RsmF consensus binding sites contribute to RsmV activity. Electrophoretic mobility shifts assays show that RsmF binds RsmV with >10-fold higher affinity than RsmY and RsmZ. Gene expression studies revealed that the temporal expression pattern of RsmV differs from RsmW, RsmY, and RsmZ. These findings suggest that each sRNA may play distinct roles in controlling RsmA and RsmF activity. IMPORTANCE The CsrA/RsmA family of RNA-binding proteins play important roles in post-transcriptional control of gene expression. The activity of CsrA/RsmA proteins is controlled by small non-coding RNAs that function as decoys to sequester CsrA/RsmA from target mRNAs. Pseudomonas aeruginosa has two CsrA family proteins (RsmA and RsmF) and at least four sequestering sRNAs (RsmV [identified in this study], RsmW, RsmY, RsmZ) that control RsmA/RsmF activity. RsmY and RsmZ are the primary sRNAs that sequester RsmA/RsmF, and RsmV and RsmW appear to play smaller roles. Differences in the temporal expression and absolute levels of the sRNAs and in their binding affinities for RsmA/RsmF may provide a mechanism of fine-tuning the output of the Rsm system in response to environmental cues. Copyright © 2018 American Society for Microbiology.

NRP-1 Receptor Expression Mismatch in Skin of Subjects with Experimental and Diabetic Small Fiber Neuropathy

PubMed Central

Van Acker, Nathalie; Ragé, Michael; Vermeirsch, Hilde; Schrijvers, Dorien; Nuydens, Rony; Byttebier, Geert; Timmers, Maarten; De Schepper, Stefanie; Streffer, Johannes; Andries, Luc; Plaghki, Léon; Cras, Patrick; Meert, Theo

2016-01-01

The in vivo cutaneous nerve regeneration model using capsaicin is applied extensively to study the regenerative mechanisms and therapeutic efficacy of disease modifying molecules for small fiber neuropathy (SFN). Since mismatches between functional and morphological nerve fiber recovery are described for this model, we aimed at determining the capability of the capsaicin model to truly mimic the morphological manifestations of SFN in diabetes. As nerve and blood vessel growth and regenerative capacities are defective in diabetes, we focused on studying the key regulator of these processes, the neuropilin-1 (NRP-1)/semaphorin pathway. This led us to the evaluation of NRP-1 receptor expression in epidermis and dermis of subjects presenting experimentally induced small fiber neuropathy, diabetic polyneuropathy and of diabetic subjects without clinical signs of small fiber neuropathy. The NRP-1 receptor was co-stained with CD31 vessel-marker using immunofluorescence and analyzed with Definiens® technology. This study indicates that capsaicin application results in significant loss of epidermal NRP-1 receptor expression, whereas diabetic subjects presenting small fiber neuropathy show full epidermal NRP-1 expression in contrast to the basal expression pattern seen in healthy controls. Capsaicin induced a decrease in dermal non-vascular NRP-1 receptor expression which did not appear in diabetic polyneuropathy. We can conclude that the capsaicin model does not mimic diabetic neuropathy related changes for cutaneous NRP-1 receptor expression. In addition, our data suggest that NRP-1 might play an important role in epidermal nerve fiber loss and/or defective regeneration and that NRP-1 receptor could change the epidermal environment to a nerve fiber repellant bed possibly through Sem3A in diabetes. PMID:27598321

Multiclass classification for skin cancer profiling based on the integration of heterogeneous gene expression series.

PubMed

Gálvez, Juan Manuel; Castillo, Daniel; Herrera, Luis Javier; San Román, Belén; Valenzuela, Olga; Ortuño, Francisco Manuel; Rojas, Ignacio

2018-01-01

Most of the research studies developed applying microarray technology to the characterization of different pathological states of any disease may fail in reaching statistically significant results. This is largely due to the small repertoire of analysed samples, and to the limitation in the number of states or pathologies usually addressed. Moreover, the influence of potential deviations on the gene expression quantification is usually disregarded. In spite of the continuous changes in omic sciences, reflected for instance in the emergence of new Next-Generation Sequencing-related technologies, the existing availability of a vast amount of gene expression microarray datasets should be properly exploited. Therefore, this work proposes a novel methodological approach involving the integration of several heterogeneous skin cancer series, and a later multiclass classifier design. This approach is thus a way to provide the clinicians with an intelligent diagnosis support tool based on the use of a robust set of selected biomarkers, which simultaneously distinguishes among different cancer-related skin states. To achieve this, a multi-platform combination of microarray datasets from Affymetrix and Illumina manufacturers was carried out. This integration is expected to strengthen the statistical robustness of the study as well as the finding of highly-reliable skin cancer biomarkers. Specifically, the designed operation pipeline has allowed the identification of a small subset of 17 differentially expressed genes (DEGs) from which to distinguish among 7 involved skin states. These genes were obtained from the assessment of a number of potential batch effects on the gene expression data. The biological interpretation of these genes was inspected in the specific literature to understand their underlying information in relation to skin cancer. Finally, in order to assess their possible effectiveness in cancer diagnosis, a cross-validation Support Vector Machines (SVM)-based classification including feature ranking was performed. The accuracy attained exceeded the 92% in overall recognition of the 7 different cancer-related skin states. The proposed integration scheme is expected to allow the co-integration with other state-of-the-art technologies such as RNA-seq.

larvalign: Aligning Gene Expression Patterns from the Larval Brain of Drosophila melanogaster.

PubMed

Muenzing, Sascha E A; Strauch, Martin; Truman, James W; Bühler, Katja; Thum, Andreas S; Merhof, Dorit

2018-01-01

The larval brain of the fruit fly Drosophila melanogaster is a small, tractable model system for neuroscience. Genes for fluorescent marker proteins can be expressed in defined, spatially restricted neuron populations. Here, we introduce the methods for 1) generating a standard template of the larval central nervous system (CNS), 2) spatial mapping of expression patterns from different larvae into a reference space defined by the standard template. We provide a manually annotated gold standard that serves for evaluation of the registration framework involved in template generation and mapping. A method for registration quality assessment enables the automatic detection of registration errors, and a semi-automatic registration method allows one to correct registrations, which is a prerequisite for a high-quality, curated database of expression patterns. All computational methods are available within the larvalign software package: https://github.com/larvalign/larvalign/releases/tag/v1.0.

Closed-form Capacity Expressions for the α-μ Fading Channel with SC Diversity under Different Adaptive Transmission Strategies

NASA Astrophysics Data System (ADS)

Mohamed, Refaat; Ismail, Mahmoud H.; Newagy, Fatma; Mourad, Heba M.

2013-03-01

Stemming from the fact that the α-μ fading distribution is one of the very general fading models used in the literature to describe the small scale fading phenomenon, in this paper, closed-form expressions for the Shannon capacity of the α-μ fading channel operating under four main adaptive transmission strategies are derived assuming integer values for μ. These expressions are derived for the case of no diversity as well as for selection combining diversity with independent and identically distributed branches. The obtained expressions reduce to those previously derived in the literature for the Weibull as well as the Rayleigh fading cases, which are both special cases of the α-μ channel. Numerical results are presented for the capacity under the four adaptive transmission strategies and the effect of the fading parameter as well as the number of diversity branches is studied.

Potential functions of microRNAs in starch metabolism and development revealed by miRNA transcriptome profiling of cassava cultivars and their wild progenitor.

PubMed

Chen, Xin; Xia, Jing; Xia, Zhiqiang; Zhang, Hefang; Zeng, Changying; Lu, Cheng; Zhang, Weixiong; Wang, Wenquan

2015-02-04

MicroRNAs (miRNAs) are small (approximately 21 nucleotide) non-coding RNAs that are key post-transcriptional gene regulators in eukaryotic organisms. More than 100 cassava miRNAs have been identified in a conservation analysis and a repertoire of cassava miRNAs have also been characterised by next-generation sequencing (NGS) in recent studies. Here, using NGS, we profiled small non-coding RNAs and mRNA genes in two cassava cultivars and their wild progenitor to identify and characterise miRNAs that are potentially involved in plant growth and starch biosynthesis. Six small RNA and six mRNA libraries from leaves and roots of the two cultivars, KU50 and Arg7, and their wild progenitor, W14, were subjected to NGS. Analysis of the sequencing data revealed 29 conserved miRNA families and 33 new miRNA families. Together, these miRNAs potentially targeted a total of 360 putative target genes. Whereas 16 miRNA families were highly expressed in cultivar leaves, another 13 miRNA families were highly expressed in storage roots of cultivars. Co-expression analysis revealed that the expression level of some targets had negative relationship with their corresponding miRNAs in storage roots and leaves; these targets included MYB33, ARF10, GRF1, RD19, APL2, NF-YA3 and SPL2, which are known to be involved in plant development, starch biosynthesis and response to environmental stimuli. The identified miRNAs, target mRNAs and target gene ontology annotation all shed light on the possible functions of miRNAs in Manihot species. The differential expression of miRNAs between cultivars and their wild progenitor, together with our analysis of GO annotation and confirmation of miRNA: target pairs, might provide insight into know the differences between wild progenitor and cultivated cassava.

Post-Transcriptional Dysregulation by miRNAs Is Implicated in the Pathogenesis of Gastrointestinal Stromal Tumor [GIST

PubMed Central

Kelly, Lorna; Bryan, Kenneth; Kim, Su Young; Janeway, Katherine A.; Killian, J. Keith; Schildhaus, Hans-Ulrich; Miettinen, Markku; Helman, Lee; Meltzer, Paul S.; van de Rijn, Matt; Debiec-Rychter, Maria; O’Sullivan, Maureen

2013-01-01

In contrast to adult mutant gastrointestinal stromal tumors [GISTs], pediatric/wild-type GISTs remain poorly understood overall, given their lack of oncogenic activating tyrosine kinase mutations. These GISTs, with a predilection for gastric origin in female patients, show limited response to therapy with tyrosine kinase inhibitors and generally pursue a more indolent course, but still may prove fatal. Defective cellular respiration appears to underpin tumor development in these wild-type cases, which as a group lack expression of succinate dehydrogenase [SDH] B, a surrogate marker for respiratory chain metabolism. Yet, only a small subset of the wild-type tumors show mutations in the genes coding for the SDH subunits [SDHx]. To explore additional pathogenetic mechanisms in these wild-type GISTs, we elected to investigate post-transcriptional regulation of these tumors by conducting microRNA (miRNA) profiling of a mixed cohort of 73 cases including 18 gastric pediatric wild-type, 25 (20 gastric, 4 small bowel and 1 retroperitoneal) adult wild-type GISTs and 30 gastric adult mutant GISTs. By this approach we have identified distinct signatures for GIST subtypes which correlate tightly with clinico-pathological parameters. A cluster of miRNAs on 14q32 show strikingly different expression patterns amongst GISTs, a finding which appears to be explained at least in part by differential allelic methylation of this imprinted region. Small bowel and retroperitoneal wild-type GISTs segregate with adult mutant GISTs and express SDHB, while adult wild-type gastric GISTs are dispersed amongst adult mutant and pediatric wild-type cases, clustering in this situation on the basis of SDHB expression. Interestingly, global methylation analysis has recently similarly demonstrated that these wild-type, SDHB-immunonegative tumors show a distinct pattern compared with KIT and PDGFRA mutant tumors, which as a rule do express SDHB. All cases with Carney triad within our cohort cluster together tightly. PMID:23717541

Post-transcriptional dysregulation by miRNAs is implicated in the pathogenesis of gastrointestinal stromal tumor [GIST].

PubMed

Kelly, Lorna; Bryan, Kenneth; Kim, Su Young; Janeway, Katherine A; Killian, J Keith; Schildhaus, Hans-Ulrich; Miettinen, Markku; Helman, Lee; Meltzer, Paul S; van de Rijn, Matt; Debiec-Rychter, Maria; O'Sullivan, Maureen

2013-01-01

In contrast to adult mutant gastrointestinal stromal tumors [GISTs], pediatric/wild-type GISTs remain poorly understood overall, given their lack of oncogenic activating tyrosine kinase mutations. These GISTs, with a predilection for gastric origin in female patients, show limited response to therapy with tyrosine kinase inhibitors and generally pursue a more indolent course, but still may prove fatal. Defective cellular respiration appears to underpin tumor development in these wild-type cases, which as a group lack expression of succinate dehydrogenase [SDH] B, a surrogate marker for respiratory chain metabolism. Yet, only a small subset of the wild-type tumors show mutations in the genes coding for the SDH subunits [SDHx]. To explore additional pathogenetic mechanisms in these wild-type GISTs, we elected to investigate post-transcriptional regulation of these tumors by conducting microRNA (miRNA) profiling of a mixed cohort of 73 cases including 18 gastric pediatric wild-type, 25 (20 gastric, 4 small bowel and 1 retroperitoneal) adult wild-type GISTs and 30 gastric adult mutant GISTs. By this approach we have identified distinct signatures for GIST subtypes which correlate tightly with clinico-pathological parameters. A cluster of miRNAs on 14q32 show strikingly different expression patterns amongst GISTs, a finding which appears to be explained at least in part by differential allelic methylation of this imprinted region. Small bowel and retroperitoneal wild-type GISTs segregate with adult mutant GISTs and express SDHB, while adult wild-type gastric GISTs are dispersed amongst adult mutant and pediatric wild-type cases, clustering in this situation on the basis of SDHB expression. Interestingly, global methylation analysis has recently similarly demonstrated that these wild-type, SDHB-immunonegative tumors show a distinct pattern compared with KIT and PDGFRA mutant tumors, which as a rule do express SDHB. All cases with Carney triad within our cohort cluster together tightly.

Dimorphic expression of sex-related genes in different gonadal development stages of sterlet, Acipenser ruthenus, a primitive fish species.

PubMed

Wang, Wei; Zhu, Hua; Dong, Ying; Tian, ZhaoHui; Dong, Tian; Hu, HongXia; Niu, CuiJuan

2017-12-01

Molecular mechanism of sex determination and differentiation of sturgeon, a primitive fish species, is extraordinarily important due to the valuable caviar; however, it is still poorly known. The present work aimed to identify the major genes involved in regulating gonadal development of sterlet, a small species of sturgeon, from 13 candidate genes which have been shown to relate to gonadal differentiation and development in other teleost fish. The sex and gonadal development of sterlets were determined by histological observation and levels of sex steroids testosterone (T), 11-ketotestosterone (11-KT), and 17β-estradiol (E2) in serum. Sexually dimorphic gene expressions were investigated. The results revealed that gonadal development were asynchronous in 2-year-old male and female sterlets with the testes in early or mid-spermatogenesis and the ovaries in chromatin nucleolus stage or perinucleolus stage, respectively. The levels of T and E2 were not significantly different between sexes or different gonadal development stages while 11-KT had the higher level in mid-spermatogenesis testis stage. In all the investigated gonadal development stages, gene dmrt1 and hsd11b2 were expressed higher in male whereas foxl2 and cyp19a1 were expressed higher in female. Thus, these genes provided the promising markers for sex identification of sterlet. It was unexpected that dkk1 and dax1 had significantly higher expression in ovarian perinucleolus stage than in ovarian chromatin nucleolus stage and in the testis, suggesting that these two genes had more correlation with ovarian development than with the testis, contrary to the previous reports in other vertebrates. Testicular development-related genes (gsdf and amh) and estrogen receptor genes (era and erb) differentially expressed at different testis or ovary development stages, but their expressions were not absolutely significantly different in male and female, depending on the gonadal development stage. Expression of androgen receptor gene ar or rspo, which was supposed to be related to ovarian development, presented no difference between gonadal development stages investigated in this study whenever in male or female.

Bioinformatic Identification of Potential MicroRNAs and Their Targets in the Lingzhi or Reishi Medicinal Mushroom Ganoderma lucidum (Higher Basidiomycetes).

PubMed

Mu, Da-Shuai; Li, Chenyang; Shi, Liang; Zhang, Xuchen; Ren, Ang; Zhao, Ming-Wen

2015-01-01

MicroRNAs (miRNAs) are a class of small, endogenous, noncoding RNA molecules that negatively regulate gene expression at the transcriptional or the post-transcriptional level. Although a large number of miRNAs have been identified in many species, especially model plants and animals, miRNAs in fungi remain largely unknown. In this study, based on a database of expressed sequence tags in Ganoderma lucidum, 89 potential miRNAs were identified using computational methods. Real-time polymerase chain reaction analysis of miRNA-like samples prepared from G. lucidum at different development stages revealed that miRNA-like RNAs were differentially expressed in different stages. Furthermore, a total of 28 potential targets were found based on near-perfect or perfect complementarity between the randomly selected 9 miRNA-like RNAs and the target sequences, and potential targets for G. lucidum miRNA-like RNAs were predicted. Finally, we studied the expression pattern of 4 target genes in 3 different development stages of G. lucidum to further understand the mechanism of interaction between miRNA-like RNAs and their target genes. Our analysis paves the way toward identifying fungal miRNA-like RNAs that might be involved in various physiological and cellular differentiation processes.

The timing and location of GDNF expression determines enteric nervous system structure and function

PubMed Central

Wang, Hongtao; Hughes, Inna; Planer, William; Parsadanian, Alexander; Grider, John R.; Vohra, Bhupinder P.S.; Keller-Peck, Cynthia; Heuckeroth, Robert O.

2010-01-01

Ret signaling is critical for formation of the enteric nervous system (ENS) because Ret activation promotes ENS precursor survival, proliferation, and migration and provides trophic support for mature enteric neurons. While these roles are well established, we now provide evidence that increasing levels of the Ret ligand GDNF in mice causes alterations in ENS structure and function that are critically dependent on the time and location of increased GDNF availability. This is demonstrated using two different strains of transgenic mice and by injecting newborn mice with GDNF. Furthermore, because different subclasses of ENS precursors withdraw from the cell cycle at different times during development, increases in GDNF at specific times alter the ratio of neuronal subclasses in the mature ENS. In addition, we confirm that esophageal neurons are GDNF responsive and demonstrate that the location of GDNF production influences neuronal process projection for NADPH diaphorase expressing, but not acetylcholinesterase, choline acetyltransferase, or tryptophan hydroxylase expressing small bowel myenteric neurons. We further demonstrate that changes in GDNF availability influence intestinal function in vitro and in vivo. Thus, changes in GDNF expression can create a wide variety of alterations in ENS structure and function and may in part contribute to human motility disorders. PMID:20107080

The timing and location of glial cell line-derived neurotrophic factor expression determine enteric nervous system structure and function.

PubMed

Wang, Hongtao; Hughes, Inna; Planer, William; Parsadanian, Alexander; Grider, John R; Vohra, Bhupinder P S; Keller-Peck, Cynthia; Heuckeroth, Robert O

2010-01-27

Ret signaling is critical for formation of the enteric nervous system (ENS) because Ret activation promotes ENS precursor survival, proliferation, and migration and provides trophic support for mature enteric neurons. Although these roles are well established, we now provide evidence that increasing levels of the Ret ligand glial cell line-derived neurotrophic factor (GDNF) in mice causes alterations in ENS structure and function that are critically dependent on the time and location of increased GDNF availability. This is demonstrated using two different strains of transgenic mice and by injecting newborn mice with GDNF. Furthermore, because different subclasses of ENS precursors withdraw from the cell cycle at different times during development, increases in GDNF at specific times alter the ratio of neuronal subclasses in the mature ENS. In addition, we confirm that esophageal neurons are GDNF responsive and demonstrate that the location of GDNF production influences neuronal process projection for NADPH diaphorase-expressing, but not acetylcholinesterase-, choline acetyltransferase-, or tryptophan hydroxylase-expressing, small bowel myenteric neurons. We further demonstrate that changes in GDNF availability influence intestinal function in vitro and in vivo. Thus, changes in GDNF expression can create a wide variety of alterations in ENS structure and function and may in part contribute to human motility disorders.

Selection for low or high primary dormancy in Lolium rigidum Gaud seeds results in constitutive differences in stress protein expression and peroxidase activity

PubMed Central

Goggin, Danica E.; Powles, Stephen B.; Steadman, Kathryn J.

2011-01-01

Seed dormancy in wild Lolium rigidum Gaud (annual ryegrass) populations is highly variable and not well characterized at the biochemical level. To identify some of the determinants of dormancy level in these seeds, the proteomes of subpopulations selected for low and high levels of primary dormancy were compared by two-dimensional polyacrylamide gel electrophoresis of extracts from mature, dry seeds. High-dormancy seeds showed higher expression of small heat shock proteins, enolase, and glyoxalase I than the low-dormancy seeds. The functional relevance of these differences in protein expression was confirmed by the fact that high-dormancy seeds were more tolerant to high temperatures imposed at imbibition and had consistently higher glyoxalase I activity over 0–42 d dark stratification. Higher expression of a putative glutathione peroxidase in low-dormancy seeds was not accompanied by higher activity, but these seeds had a slightly more oxidized glutathione pool and higher total peroxidase activity. Overall, these biochemical and physiological differences suggest that L. rigidum seeds selected for low dormancy are more prepared for rapid germination via peroxidase-mediated cell wall weakening, whilst seeds selected for high dormancy are constitutively prepared to survive environmental stresses, even in the absence of stress during seed development. PMID:20974739

[Clinical significance and mechanism of upregulation of PI3Kp110α in non-small cell lung carcinoma].

PubMed

Xiong, Y; Qu, L L; Li, D; Wang, Y; Li, T

2017-10-23

Objective: To investigate the clinical significance and mechanism of upregulation of phosphoinositide 3-kinase p110α(PI3Kp110α)in non-small cell lung carcinoma (NSCLC). Methods: Expressions of PI3Kp110α and other components in PI3K signaling pathway (including phospho-Akt (p-Akt, Ser 473), MET, ROS1, HER-2, ALK, total EGFR and mutant EGFR) and p53 (the transcription factor of PIK3CA) mutation in NSCLC were detected by immunohistochemistry. The relationships between PI3Kp110α expression and clinicopathological characteristics, expressions of other proteins in PI3K pathway and p53 mutation were analyzed. Results: In 170 NSCLC patients, 72 cases (42.4%) showed lower expression and 98 cases (57.6%) showed higher expression of PI3Kp110α. Upregulation of PI3Kp110α was not significantly associated with gender, age, T stage and pathologic grade ( P >0.05). While upregulation of PI3Kp110α was significantly associated with smoking status of patients, pathologic classification, N stage, TNM stage and Ki-67 index ( P <0.05). Expression of PI3Kp110α was positively correlated with expressions of MET ( P <0.05) and mutant EGFR ( P =0.018), while not significantly related with expressions of p-Akt(Ser473), HER-2, ALK, ROS1, total EGFR or p53 mutation ( P >0.05). Conclusions: Upregulation of PI3Kp110α is closely related with tumorigenesis of non-smoking lung adenocarcinoma. MET overexpression and EGFR mutation may be crucial to upregulate expression of PI3Kp110α in NSCLC. Overexpression of PI3Kp110α may inhibit tumor cell proliferation in NSCLC through a different pathway other than classical PI3K pathway. Upregulation of PI3Kp110α may predict favorable prognosis of NSCLC patients.

Excess fertilizer responsive miRNAs revealed in Linum usitatissimum L.

PubMed

Melnikova, Nataliya V; Dmitriev, Alexey A; Belenikin, Maxim S; Speranskaya, Anna S; Krinitsina, Anastasia A; Rachinskaia, Olga A; Lakunina, Valentina A; Krasnov, George S; Snezhkina, Anastasiya V; Sadritdinova, Asiya F; Uroshlev, Leonid A; Koroban, Nadezda V; Samatadze, Tatiana E; Amosova, Alexandra V; Zelenin, Alexander V; Muravenko, Olga V; Bolsheva, Nadezhda L; Kudryavtseva, Anna V

2015-02-01

Effective fertilizer application is necessary to increase crop yields and reduce risk of plant overdosing. It is known that expression level of microRNAs (miRNAs) alters in plants under different nutrient concentrations in soil. The aim of our study was to identify and characterize miRNAs with expression alterations under excessive fertilizer in agriculturally important crop - flax (Linum usitatissimum L.). We have sequenced small RNAs in flax grown under normal and excessive fertilizer using Illumina GAIIx. Over 14 million raw reads was obtained for two small RNA libraries. 84 conserved miRNAs from 20 families were identified. Differential expression was revealed for several flax miRNAs under excessive fertilizer according to high-throughput sequencing data. For 6 miRNA families (miR395, miR169, miR408, miR399, miR398 and miR168) expression level alterations were evaluated on the extended sampling using qPCR. Statistically significant up-regulation was revealed for miR395 under excessive fertilizer. It is known that target genes of miR395 are involved in sulfate uptake and assimilation. However, according to our data alterations of the expression level of miR395 could be associated not only with excess sulfur application, but also with redundancy of other macro- and micronutrients. Furthermore expression level was evaluated for miRNAs and their predicted targets. The negative correlation between miR399 expression and expression of its predicted target ubiquitin-conjugating enzyme E2 gene was shown in flax for the first time. So we suggested miR399 involvement in phosphate regulation in L. usitatissimum. Revealed in our study expression alterations contribute to miRNA role in flax response to excessive fertilizer. Copyright © 2014 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.

Sex specific expression and distribution of small RNAs in papaya.

PubMed

Aryal, Rishi; Jagadeeswaran, Guru; Zheng, Yun; Yu, Qingyi; Sunkar, Ramanjulu; Ming, Ray

2014-01-13

Regulatory function of small non-coding RNAs (sRNA) in response to environmental and developmental cues has been established. Additionally, sRNA, also plays an important role in maintaining the heterochromatin and centromere structures of the chromosome. Papaya, a trioecious species with recently evolved sex chromosomes, has emerged as an excellent model system to study sex determination and sex chromosome evolution in plants. However, role of small RNA in papaya sex determination is yet to be explored. We analyzed the high throughput sRNAs reads in the Illumina libraries prepared from male, female, and hermaphrodite flowers of papaya. Using the sRNA reads, we identified 29 miRNAs that were not previously reported from papaya. Including this and two previous studies, a total of 90 miRNAs has been identified in papaya. We analyzed the expression of these miRNAs in each sex types. A total of 65 miRNAs, including 31 conserved and 34 novel mirNA, were detected in at least one library. Fourteen of the 65 miRNAs were differentially expressed among different sex types. Most of the miRNA expressed higher in male flowers were related to the auxin signaling pathways, whereas the miRNAs expressed higher in female flowers were the potential regulators of the apical meristem identity genes. Aligning the sRNA reads identified the sRNA hotspots adjacent to the gaps of the X and Y chromosomes. The X and Y chromosomes sRNA hotspots has a 7.8 and 4.4 folds higher expression of sRNA, respectively, relative to the chromosome wide average. Approximately 75% of the reads aligned to the X chromosome hotspot was identical to that of the Y chromosome hotspot. By analyzing the large-scale sRNA sequences from three sex types, we identified the sRNA hotspots flanking the gaps of papaya X, Y, and Yh chromosome. The sRNAs expression patterns in these regions were reminiscent of the pericentromeric region indicating that the only remaining gap in each of these chromosomes is likely the centromere. We also identified 14 differentially expressed miRNAs in male, female and hermaphrodite flowers of papaya. Our results provide valuable information toward understanding the papaya sex determination.

Expression, Distribution and Role of Aquaporin Water Channels in Human and Animal Stomach and Intestines

PubMed Central

Zhu, Cui; Chen, Zhuang; Jiang, Zongyong

2016-01-01

Stomach and intestines are involved in the secretion of gastrointestinal fluids and the absorption of nutrients and fluids, which ensure normal gut functions. Aquaporin water channels (AQPs) represent a major transcellular route for water transport in the gastrointestinal tract. Until now, at least 11 AQPs (AQP1–11) have been found to be present in the stomach, small and large intestines. These AQPs are distributed in different cell types in the stomach and intestines, including gastric epithelial cells, gastric glands cells, absorptive epithelial cells (enterocytes), goblet cells and Paneth cells. AQP1 is abundantly distributed in the endothelial cells of the gastrointestinal tract. AQP3 and AQP4 are mainly distributed in the basolateral membrane of epithelial cells in the stomach and intestines. AQP7, AQP8, AQP10 and AQP11 are distributed in the apical of enterocytes in the small and large intestines. Although AQP-null mice displayed almost no phenotypes in gastrointestinal tracts, the alterations of the expression and localization of these AQPs have been shown to be associated with the pathology of gastrointestinal disorders, which suggests that AQPs play important roles serving as potential therapeutic targets. Therefore, this review provides an overview of the expression, localization and distribution of AQPs in the stomach, small and large intestine of human and animals. Furthermore, this review emphasizes the potential roles of AQPs in the physiology and pathophysiology of stomach and intestines. PMID:27589719

Expression, Distribution and Role of Aquaporin Water Channels in Human and Animal Stomach and Intestines.

PubMed

Zhu, Cui; Chen, Zhuang; Jiang, Zongyong

2016-08-29

Stomach and intestines are involved in the secretion of gastrointestinal fluids and the absorption of nutrients and fluids, which ensure normal gut functions. Aquaporin water channels (AQPs) represent a major transcellular route for water transport in the gastrointestinal tract. Until now, at least 11 AQPs (AQP1-11) have been found to be present in the stomach, small and large intestines. These AQPs are distributed in different cell types in the stomach and intestines, including gastric epithelial cells, gastric glands cells, absorptive epithelial cells (enterocytes), goblet cells and Paneth cells. AQP1 is abundantly distributed in the endothelial cells of the gastrointestinal tract. AQP3 and AQP4 are mainly distributed in the basolateral membrane of epithelial cells in the stomach and intestines. AQP7, AQP8, AQP10 and AQP11 are distributed in the apical of enterocytes in the small and large intestines. Although AQP-null mice displayed almost no phenotypes in gastrointestinal tracts, the alterations of the expression and localization of these AQPs have been shown to be associated with the pathology of gastrointestinal disorders, which suggests that AQPs play important roles serving as potential therapeutic targets. Therefore, this review provides an overview of the expression, localization and distribution of AQPs in the stomach, small and large intestine of human and animals. Furthermore, this review emphasizes the potential roles of AQPs in the physiology and pathophysiology of stomach and intestines.

Def-6, a novel regulator of small GTPases in podocytes, acts downstream of atypical protein kinase C (aPKC) λ/ι.

PubMed

Worthmann, Kirstin; Leitges, Michael; Teng, Beina; Sestu, Marcello; Tossidou, Irini; Samson, Thomas; Haller, Hermann; Huber, Tobias B; Schiffer, Mario

2013-12-01

The atypical protein kinase C (aPKC) isotypes PKCλ/ι and PKCζ are both expressed in podocytes; however, little is known about differences in their function. Previous studies in mice have demonstrated that podocyte-specific loss of PKCλ/ι leads to a severe glomerular phenotype, whereas mice deficient in PKCζ develop no renal phenotype. We analyzed various effects caused by PKCλ/ι and PKCζ deficiency in cultured murine podocytes. In contrast to PKCζ-deficient podocytes, PKCλ/ι-deficient podocytes exhibited a severe actin cytoskeletal phenotype, reduced cell size, decreased number of focal adhesions, and increased activation of small GTPases. Comparative microarray analysis revealed that the guanine nucleotide exchange factor Def-6 was specifically up-regulated in PKCλ/ι-deficient podocytes. In vivo Def-6 expression is significantly increased in podocytes of PKCλ/ι-deficient mice. Cultured PKCλ/ι-deficient podocytes exhibited an enhanced membrane association of Def-6, indicating enhanced activation. Overexpression of aPKCλ/ι in PKCλ/ι-deficient podocytes could reduce the membrane-associated expression of Def-6 and rescue the actin phenotype. In the present study, PKCλ/ι was identified as an important factor for actin cytoskeletal regulation in podocytes and Def-6 as a specific downstream target of PKCλ/ι that regulates the activity of small GTPases and subsequently the actin cytoskeleton of podocytes. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Genome-Wide Identification and Comparative Analysis of Conserved and Novel MicroRNAs in Grafted Watermelon by High-Throughput Sequencing

PubMed Central

Liu, Na; Yang, Jinghua; Guo, Shaogui; Xu, Yong; Zhang, Mingfang

2013-01-01

MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs involved in the post-transcriptional gene regulation and play a critical role in plant growth, development and stresses response. However less is known about miRNAs involvement in grafting behaviors, especially with the watermelon (Citrullus lanatus L.) crop, which is one of the most important agricultural crops worldwide. Grafting method is commonly used in watermelon production in attempts to improve its adaptation to abiotic and biotic stresses, in particular to the soil-borne fusarium wilt disease. In this study, Solexa sequencing has been used to discover small RNA populations and compare miRNAs on genome-wide scale in watermelon grafting system. A total of 11,458,476, 11,614,094 and 9,339,089 raw reads representing 2,957,751, 2,880,328 and 2,964,990 unique sequences were obtained from the scions of self-grafted watermelon and watermelon grafted on-to bottle gourd and squash at two true-leaf stage, respectively. 39 known miRNAs belonging to 30 miRNA families and 80 novel miRNAs were identified in our small RNA dataset. Compared with self-grafted watermelon, 20 (5 known miRNA families and 15 novel miRNAs) and 47 (17 known miRNA families and 30 novel miRNAs) miRNAs were expressed significantly different in watermelon grafted on to bottle gourd and squash, respectively. MiRNAs expressed differentially when watermelon was grafted onto different rootstocks, suggesting that miRNAs might play an important role in diverse biological and metabolic processes in watermelon and grafting may possibly by changing miRNAs expressions to regulate plant growth and development as well as adaptation to stresses. The small RNA transcriptomes obtained in this study provided insights into molecular aspects of miRNA-mediated regulation in grafted watermelon. Obviously, this result would provide a basis for further unravelling the mechanism on how miRNAs information is exchanged between scion and rootstock in grafted watermelon, and its relevance to diverse biological processes and environmental adaptation. PMID:23468976

DOE Office of Scientific and Technical Information (OSTI.GOV)

Poulsen, Sarah S., E-mail: spo@nrcwe.dk; Department of Science, Systems and Models, Roskilde University, DK-4000 Roskilde; Saber, Anne T., E-mail: ats@nrcwe.dk

Multi-walled carbon nanotubes (MWCNTs) are an inhomogeneous group of nanomaterials that vary in lengths, shapes and types of metal contamination, which makes hazard evaluation difficult. Here we present a toxicogenomic analysis of female C57BL/6 mouse lungs following a single intratracheal instillation of 0, 18, 54 or 162 μg/mouse of a small, curled (CNT{sub Small}, 0.8 ± 0.1 μm in length) or large, thick MWCNT (CNT{sub Large}, 4 ± 0.4 μm in length). The two MWCNTs were extensively characterized by SEM and TEM imaging, thermogravimetric analysis, and Brunauer–Emmett–Teller surface area analysis. Lung tissues were harvested 24 h, 3 days and 28more » days post-exposure. DNA microarrays were used to analyze gene expression, in parallel with analysis of bronchoalveolar lavage fluid, lung histology, DNA damage (comet assay) and the presence of reactive oxygen species (dichlorodihydrofluorescein assay), to profile and characterize related pulmonary endpoints. Overall changes in global transcription following exposure to CNT{sub Small} or CNT{sub Large} were similar. Both MWCNTs elicited strong acute phase and inflammatory responses that peaked at day 3, persisted up to 28 days, and were characterized by increased cellular influx in bronchoalveolar lavage fluid, interstitial pneumonia and gene expression changes. However, CNT{sub Large} elicited an earlier onset of inflammation and DNA damage, and induced more fibrosis and a unique fibrotic gene expression signature at day 28, compared to CNT{sub Small}. The results indicate that the extent of change at the molecular level during early response phases following an acute exposure is greater in mice exposed to CNT{sub Large}, which may eventually lead to the different responses observed at day 28. - Highlights: • We evaluate the toxicogenomic response in mice following MWCNT instillation. • Two MWCNTs of different properties were examined and thoroughly characterized. • MWCNT exposure leads to increased pulmonary inflammation and acute phase response. • The thick and straight MWCNT induced transcriptional and histological pulmonary fibrotic changes. • This was not observed following exposure to the thinner and curled MWCNT.« less

The role of TRPV1 in different subtypes of dorsal root ganglion neurons in rat chronic inflammatory nociception induced by complete Freund's adjuvant

PubMed Central

Yu, Lu; Yang, Fei; Luo, Hao; Liu, Feng-Yu; Han, Ji-Sheng; Xing, Guo-Gang; Wan, You

2008-01-01

Background The present study aims to investigate the role of transient receptor potential vanilloid 1 (TRPV1) in dorsal root ganglion (DRG) neurons in chronic pain including thermal hyperalgesia and mechanical allodynia. Chronic inflammatory nociception of rats was produced by intraplantar injection of complete Freund's adjuvant (CFA) and data was collected until day 28 following injection. Results Thermal hyperalgesia was evident from day 1 to day 28 with peak at day 7, while mechanical allodynia persisted from day 1 to day 14 and was greatest at day 7. Intrathecal administration of AMG 9810 at day 7, a selective TRPV1 antagonist, significantly reduced thermal hyperalgesia and mechanical allodynia. TRPV1 expression in DRG detected by Western blotting was increased relative to baseline throughout the observation period. Double labeling of TRPV1 with neuronal marker neurofilament 200 (NF200), calcitonin gene-related peptide (CGRP) or isolectin B4 (IB4) was used to distinguish different subtypes of DRG neurons. TRPV1 expression was increased in the medium-sized myelinated A fiber (NF200 positive) neurons and in small non-peptidergic (IB4 positive) neurons from day 1 to day 14 and was increased in small peptidergic (CGRP positive) neurons from day 1 to day 28. Conclusion TRPV1 expression increases in all three types of DRG neurons after CFA injection and plays a role in CFA-induced chronic inflammatory pain including thermal hyperalgesia and mechanical allodynia. PMID:19055783

Up-regulation of heat shock proteins is essential for cold survival during insect diapause

PubMed Central

Rinehart, Joseph P.; Li, Aiqing; Yocum, George D.; Robich, Rebecca M.; Hayward, Scott A. L.; Denlinger, David L.

2007-01-01

Diapause, the dormancy common to overwintering insects, evokes a unique pattern of gene expression. In the flesh fly, most, but not all, of the fly's heat shock proteins (Hsps) are up-regulated. The diapause up-regulated Hsps include two members of the Hsp70 family, one member of the Hsp60 family (TCP-1), at least four members of the small Hsp family, and a small Hsp pseudogene. Expression of an Hsp70 cognate, Hsc70, is uninfluenced by diapause, and Hsp90 is actually down-regulated during diapause, thus diapause differs from common stress responses that elicit synchronous up-regulation of all Hsps. Up-regulation of the Hsps begins at the onset of diapause, persists throughout the overwintering period, and ceases within hours after the fly receives the signal to reinitiate development. The up-regulation of Hsps appears to be common to diapause in species representing diverse insect orders including Diptera, Lepidoptera, Coleoptera, and Hymenoptera as well as in diapauses that occur in different developmental stages (embryo, larva, pupa, adult). Suppressing expression of Hsp23 and Hsp70 in flies by using RNAi did not alter the decision to enter diapause or the duration of diapause, but it had a profound effect on the pupa's ability to survive low temperatures. We thus propose that up-regulation of Hsps during diapause is a major factor contributing to cold-hardiness of overwintering insects. PMID:17522254

Flow cytometric characterization of culture expanded multipotent mesenchymal stromal cells (MSCs) from horse adipose tissue: towards the definition of minimal stemness criteria.

PubMed

Pascucci, L; Curina, G; Mercati, F; Marini, C; Dall'Aglio, C; Paternesi, B; Ceccarelli, P

2011-12-15

In the last decades, multipotent mesenchymal progenitor cells have been isolated from many adult tissues of different species. The International Society for Cellular Therapy (ISCT) has recently established that multipotent mesenchymal stromal cells (MSCs) is the currently recommended designation. In this study, we used flow cytometry to evaluate the expression of several molecules related to stemness (CD90, CD44, CD73 and STRO-1) in undifferentiated, early-passaged MSCs isolated from adipose tissue of four donor horses (AdMSCs). The four populations unanimously expressed high levels of CD90 and CD44. On the contrary, they were unexpectedly negative to CD73. A small percentage of the cells, finally, showed the expression of STRO-1. This last result might be due to the existence of a small subpopulation of STRO-1+ cells or to a poor cross-reactivity of the antibody. A remarkable donor-to-donor consistency and reproducibility of these findings was demonstrated. The data presented herein support the idea that equine AdMSCs may be easily isolated and selected by adherence to tissue culture plastic and exhibit a surface profile characterized by some peculiar differences in comparison to those described in other species. Continued characterization of these cells will help to clarify several aspects of their biology and may ultimately enable the isolation of specific, purified subpopulations. Copyright © 2011 Elsevier B.V. All rights reserved.

Small Systems and Limitations on the Use of Chemical Thermodynamics

NASA Astrophysics Data System (ADS)

Tovbin, Yu. K.

2018-01-01

Limitations on using chemical thermodynamics to describe small systems are formulated. These limitations follow from statistical mechanics for equilibrium and nonequilibrium processes and reflect (1) differences between characteristic relaxation times in momentum, energy, and mass transfer in different aggregate states of investigated systems; (2) achievements of statistical mechanics that allow us to determine criteria for the size of smallest region in which thermodynamics can be applied and the scale of the emergence of a new phase, along with criteria for the conditions of violating a local equilibrium. Based on this analysis, the main thermodynamic results are clarified: the phase rule for distorted interfaces, the sense and area of applicability of Gibbs's concept of passive forces, and the artificiality of Kelvin's equation as a result of limitations on the thermodynamic approach to considering small bodies. The wrongness of introducing molecular parameters into thermodynamic derivations, and the activity coefficient for an activated complex into the expression for a reaction rate constant, is demonstrated.

Target engagement imaging of PARP inhibitors in small-cell lung cancer.

PubMed

Carney, Brandon; Kossatz, Susanne; Lok, Benjamin H; Schneeberger, Valentina; Gangangari, Kishore K; Pillarsetty, Naga Vara Kishore; Weber, Wolfgang A; Rudin, Charles M; Poirier, John T; Reiner, Thomas

2018-01-12

Insufficient chemotherapy response and rapid disease progression remain concerns for small-cell lung cancer (SCLC). Oncologists rely on serial CT scanning to guide treatment decisions, but this cannot assess in vivo target engagement of therapeutic agents. Biomarker assessments in biopsy material do not assess contemporaneous target expression, intratumoral drug exposure, or drug-target engagement. Here, we report the use of PARP1/2-targeted imaging to measure target engagement of PARP inhibitors in vivo. Using a panel of clinical PARP inhibitors, we show that PARP imaging can quantify target engagement of chemically diverse small molecule inhibitors in vitro and in vivo. We measure PARP1/2 inhibition over time to calculate effective doses for individual drugs. Using patient-derived xenografts, we demonstrate that different therapeutics achieve similar integrated inhibition efficiencies under different dosing regimens. This imaging approach to non-invasive, quantitative assessment of dynamic intratumoral target inhibition may improve patient care through real-time monitoring of drug delivery.

Psmir: a database of potential associations between small molecules and miRNAs

PubMed Central

Meng, Fanlin; Wang, Jing; Dai, Enyu; Yang, Feng; Chen, Xiaowen; Wang, Shuyuan; Yu, Xuexin; Liu, Dianming; Jiang, Wei

2016-01-01

miRNAs are key post-transcriptional regulators of many essential biological processes, and their dysregulation has been validated in almost all human cancers. Restoring aberrantly expressed miRNAs might be a novel therapeutics. Recently, many studies have demonstrated that small molecular compounds can affect miRNA expression. Thus, prediction of associations between small molecules and miRNAs is important for investigation of miRNA-targeted drugs. Here, we analyzed 39 miRNA-perturbed gene expression profiles, and then calculated the similarity of transcription responses between miRNA perturbation and drug treatment to predict drug-miRNA associations. At the significance level of 0.05, we obtained 6501 candidate associations between 1295 small molecules and 25 miRNAs, which included 624 FDA approved drugs. Finally, we constructed the Psmir database to store all potential associations and the related materials. In a word, Psmir served as a valuable resource for dissecting the biological significance in small molecules’ effects on miRNA expression, which will facilitate developing novel potential therapeutic targets or treatments for human cancers. Psmir is supported by all major browsers, and is freely available at http://www.bio-bigdata.com/Psmir/. PMID:26759061

Psmir: a database of potential associations between small molecules and miRNAs.

PubMed

Meng, Fanlin; Wang, Jing; Dai, Enyu; Yang, Feng; Chen, Xiaowen; Wang, Shuyuan; Yu, Xuexin; Liu, Dianming; Jiang, Wei

2016-01-13

miRNAs are key post-transcriptional regulators of many essential biological processes, and their dysregulation has been validated in almost all human cancers. Restoring aberrantly expressed miRNAs might be a novel therapeutics. Recently, many studies have demonstrated that small molecular compounds can affect miRNA expression. Thus, prediction of associations between small molecules and miRNAs is important for investigation of miRNA-targeted drugs. Here, we analyzed 39 miRNA-perturbed gene expression profiles, and then calculated the similarity of transcription responses between miRNA perturbation and drug treatment to predict drug-miRNA associations. At the significance level of 0.05, we obtained 6501 candidate associations between 1295 small molecules and 25 miRNAs, which included 624 FDA approved drugs. Finally, we constructed the Psmir database to store all potential associations and the related materials. In a word, Psmir served as a valuable resource for dissecting the biological significance in small molecules' effects on miRNA expression, which will facilitate developing novel potential therapeutic targets or treatments for human cancers. Psmir is supported by all major browsers, and is freely available at http://www.bio-bigdata.com/Psmir/.

The significance of PIWI family expression in human lung embryogenesis and non-small cell lung cancer

PubMed Central

Navarro, Alfons; Tejero, Rut; Viñolas, Nuria; Cordeiro, Anna; Marrades, Ramon M.; Fuster, Dolors; Caritg, Oriol; Moises, Jorge; Muñoz, Carmen; Molins, Laureano; Ramirez, Josep; Monzo, Mariano

2015-01-01

The expression of Piwi-interacting RNAs, small RNAs that bind to PIWI proteins, was until recently believed to be limited to germinal stem cells. We have studied the expression of PIWI genes during human lung embryogenesis and in paired tumor and normal tissue prospectively collected from 71 resected non-small-cell lung cancer patients. The mRNA expression analysis showed that PIWIL1 was highly expressed in 7-week embryos and downregulated during the subsequent weeks of development. PIWIL1 was expressed in 11 of the tumor samples but in none of the normal tissue samples. These results were validated by immunohistochemistry, showing faint cytoplasmic reactivity in the PIWIL1-positive samples. Interestingly, the patients expressing PIWIL1 had a shorter time to relapse (TTR) (p = 0.006) and overall survival (OS) (p = 0.0076) than those without PIWIL1 expression. PIWIL2 and 4 were downregulated in tumor tissue in comparison to the normal tissue (p < 0.001) and the patients with lower levels of PIWIL4 had shorter TTR (p = 0.048) and OS (p = 0.033). In the multivariate analysis, PIWIL1 expression emerged as an independent prognostic marker. Using 5-Aza-dC treatment and bisulfite sequencing, we observed that PIWIL1 expression could be regulated in part by methylation. Finally, an in silico study identified a stem-cell expression signature associated with PIWIL1 expression. PMID:25742785

Variation in flooding-induced morphological traits in natural populations of white clover (Trifolium repens) and their effects on plant performance during soil flooding

PubMed Central

Huber, Heidrun; Jacobs, Elke; Visser, Eric J. W.

2009-01-01

Background and Aims Soil flooding leads to low soil oxygen concentrations and thereby negatively affects plant growth. Differences in flooding tolerance have been explained by the variation among species in the extent to which traits related to acclimation were expressed. However, our knowledge of variation within natural species (i.e. among individual genotypes) in traits related to flooding tolerance is very limited. Such data could tell us on which traits selection might have taken place, and will take place in future. The aim of the present study was to show that variation in flooding-tolerance-related traits is present among genotypes of the same species, and that both the constitutive variation and the plastic variation in flooding-induced changes in trait expression affect the performance of genotypes during soil flooding. Methods Clones of Trifolium repens originating from a river foreland were subjected to either drained, control conditions or to soil flooding. Constitutive expression of morphological traits was recorded on control plants, and flooding-induced changes in expression were compared with these constitutive expression levels. Moreover, the effect of both constitutive and flooding-induced trait expression on plant performance was determined. Key Results Constitutive and plastic variation of several morphological traits significantly affected plant performance. Even relatively small increases in root porosity and petiole length contributed to better performance during soil flooding. High specific leaf area, by contrast, was negatively correlated with performance during flooding. Conclusions The data show that different genotypes responded differently to soil flooding, which could be linked to variation in morphological trait expression. As flooded and drained conditions exerted different selection pressures on trait expression, the optimal value for constitutive and plastic traits will depend on the frequency and duration of flooding. These data will help us understanding the mechanisms affecting short- and long-term dynamics in flooding-prone ecosystems. PMID:18713824

Gene expression signatures in tree shrew choroid in response to three myopiagenic conditions

PubMed Central

He, Li; Frost, Michael R.; Siegwart, John T.; Norton, Thomas T.

2014-01-01

We examined gene expression in tree shrew choroid in response to three different myopiagenic conditions: minus lens (ML) wear, form deprivation (FD), and continuous darkness (DK). Four groups of tree shrews (n = 7 per group) were used. Starting 24 days after normal eye opening (days of visual experience [DVE]), the ML group wore a monocular −5 D lens for 2 days. The FD group wore a monocular translucent diffuser for 2 days. The DK group experienced continuous darkness binocularly for 11 days, starting at 17 DVE. An age-matched normal group was examined at 26 DVE. Quantitative PCR was used to measure the relative (treated eye vs. control eye) differences in mRNA levels in the choroid for 77 candidate genes. Small myopic changes were observed in the treated eyes (relative to the control eyes) of the ML group (−1.0 ± 0.2 D; mean ± SEM) and FD group (−1.9 ± 0.2 D). A larger myopia developed in the DK group (−4.4 ± 1.0 D) relative to Normal eyes (both groups, mean of right and left eyes). In the ML group, 28 genes showed significant differential mRNA expression; eighteen were down-regulated. A very similar pattern occurred in the FD group; twenty-seven of the same genes were similarly regulated, along with five additional genes. Fewer expression differences in the DK group were significant compared to normal or the control eyes of the ML and FD groups, but the pattern was similar to that of the ML and FD differential expression patterns. These data suggest that, at the level of the choroid, the gene expression signatures produced by “GO” emmetropization signals are highly similar despite the different visual conditions. PMID:25072854

Systematic approach identifies RHOA as a potential biomarker therapeutic target for Asian gastric cancer

PubMed Central

Jung, Hae Rim; Park, Hee Seo; Park, Sungjin; Ahn, Young Zoo; Huh, Iksoo; Balch, Curt; Ku, Ja-Lok; Powis, Garth; Park, Taesung; Jeong, Jin-Hyun; Kim, Yon Hui

2016-01-01

Gastric cancer (GC) is a highly heterogeneous disease, in dire need of specific, biomarker-driven cancer therapies. While the accumulation of cancer “Big Data” has propelled the search for novel molecular targets for GC, its specific subpathway and cellular functions vary from patient to patient. In particular, mutations in the small GTPase gene RHOA have been identified in recent genome-wide sequencing of GC tumors. Moreover, protein overexpression of RHOA was reported in Chinese populations, while RHOA mutations were found in Caucasian GC tumors. To develop evidence-based precision medicine for heterogeneous cancers, we established a systematic approach to integrate transcriptomic and genomic data. Predicted signaling subpathways were then laboratory-validated both in vitro and in vivo, resulting in the identification of new candidate therapeutic targets. Here, we show: i) differences in RHOA expression patterns, and its pathway activity, between Asian and Caucasian GC tumors; ii) in vitro and in vivo perturbed RHOA expression inhibits GC cell growth in high RHOA-expressing cell lines; iii) inverse correlation between RHOA and RHOB expression; and iv) an innovative small molecule design strategy for RHOA inhibitors. In summary, RHOA, and its oncogenic signaling pathway, represent a strong biomarker-driven therapeutic target for Asian GC. This comprehensive strategy represents a promising approach for the development of “hit” compounds. PMID:27806312

Genome-wide profiling of the PIWI-interacting RNA-mRNA regulatory networks in epithelial ovarian cancers.

PubMed

Singh, Garima; Roy, Jyoti; Rout, Pratiti; Mallick, Bibekanand

2018-01-01

PIWI-interacting (piRNAs), ~23-36 nucleotide-long small non-coding RNAs (sncRNAs), earlier believed to be germline-specific, have now been identified in somatic cells, including cancer cells. These sncRNAs impact critical biological processes by fine-tuning gene expression at post-transcriptional and epigenetic levels. The expression of piRNAs in ovarian cancer, the most lethal gynecologic cancer is largely uncharted. In this study, we investigated the expression of PIWILs by qRT-PCR and western blotting and then identified piRNA transcriptomes in tissues of normal ovary and two most prevalent epithelial ovarian cancer subtypes, serous and endometrioid by small RNA sequencing. We detected 219, 256 and 234 piRNAs in normal ovary, endometrioid and serous ovarian cancer samples respectively. We observed piRNAs are encoded from various genomic regions, among which introns harbor the majority of them. Surprisingly, piRNAs originated from different genomic contexts showed the varied level of conservations across vertebrates. The functional analysis of predicted targets of differentially expressed piRNAs revealed these could modulate key processes and pathways involved in ovarian oncogenesis. Our study provides the first comprehensive piRNA landscape in these samples and a useful resource for further functional studies to decipher new mechanistic views of piRNA-mediated gene regulatory networks affecting ovarian oncogenesis. The RNA-seq data is submitted to GEO database (GSE83794).

The effect of red light and far-red light conditions on secondary metabolism in agarwood.

PubMed

Kuo, Tony Chien-Yen; Chen, Chuan-Hung; Chen, Shu-Hwa; Lu, I-Hsuan; Chu, Mei-Ju; Huang, Li-Chun; Lin, Chung-Yen; Chen, Chien-Yu; Lo, Hsiao-Feng; Jeng, Shih-Tong; Chen, Long-Fang O

2015-06-12

Agarwood, a heartwood derived from Aquilaria trees, is a valuable commodity that has seen prevalent use among many cultures. In particular, it is widely used in herbal medicine and many compounds in agarwood are known to exhibit medicinal properties. Although there exists much research into medicinal herbs and extraction of high value compounds, few have focused on increasing the quantity of target compounds through stimulation of its related pathways in this species. In this study, we observed that cucurbitacin yield can be increased through the use of different light conditions to stimulate related pathways and conducted three types of high-throughput sequencing experiments in order to study the effect of light conditions on secondary metabolism in agarwood. We constructed genome-wide profiles of RNA expression, small RNA, and DNA methylation under red light and far-red light conditions. With these profiles, we identified a set of small RNA which potentially regulates gene expression via the RNA-directed DNA methylation pathway. We demonstrate that light conditions can be used to stimulate pathways related to secondary metabolism, increasing the yield of cucurbitacins. The genome-wide expression and methylation profiles from our study provide insight into the effect of light on gene expression for secondary metabolism in agarwood and provide compelling new candidates towards the study of functional secondary metabolic components.

Abcb1 in Pigs: Molecular cloning, tissues distribution, functional analysis, and its effect on pharmacokinetics of enrofloxacin

PubMed Central

Guo, Tingting; Huang, Jinhu; Zhang, Hongyu; Dong, Lingling; Guo, Dawei; Guo, Li; He, Fang; Bhutto, Zohaib Ahmed; Wang, Liping

2016-01-01

P-glycoprotein (P-gp) is one of the best-known ATP-dependent efflux transporters, contributing to differences in pharmacokinetics and drug-drug interactions. Until now, studies on pig P-gp have been scarce. In our studies, the full-length porcine P-gp cDNA was cloned and expressed in a Madin-Darby Canine Kidney (MDCK) cell line. P-gp expression was then determined in tissues and its role in the pharmacokinetics of oral enrofloxacin in pigs was studied. The coding region of pig Abcb1 gene was 3,861 bp, encoding 1,286 amino acid residues (Mw = 141,966). Phylogenetic analysis indicated a close evolutionary relationship between porcine P-gp and those of cow and sheep. Pig P-gp was successfully stably overexpressed in MDCK cells and had efflux activity for rhodamine 123, a substrate of P-gp. Tissue distribution analysis indicated that P-gp was highly expressed in brain capillaries, small intestine, and liver. In MDCK-pAbcb1 cells, enrofloxacin was transported by P-gp with net efflux ratio of 2.48 and the efflux function was blocked by P-gp inhibitor verapamil. High expression of P-gp in the small intestine could modify the pharmacokinetics of orally administrated enrofloxacin by increasing the Cmax, AUC and Ka, which was demonstrated using verapamil, an inhibitor of P-gp. PMID:27572343

Transcriptional profile of fibroblasts obtained from the primary site, lymph node and bone marrow of breast cancer patients.

PubMed

Del Valle, Paulo Roberto; Milani, Cintia; Brentani, Maria Mitzi; Katayama, Maria Lucia Hirata; de Lyra, Eduardo Carneiro; Carraro, Dirce Maria; Brentani, Helena; Puga, Renato; Lima, Leandro A; Rozenchan, Patricia Bortman; Nunes, Bárbara Dos Santos; Góes, João Carlos Guedes Sampaio; Azevedo Koike Folgueira, Maria Aparecida

2014-09-01

Cancer-associated fibroblasts (CAF) influence tumor development at primary as well as in metastatic sites, but there have been no direct comparisons of the transcriptional profiles of stromal cells from different tumor sites. In this study, we used customized cDNA microarrays to compare the gene expression profile of stromal cells from primary tumor (CAF, n = 4), lymph node metastasis (N+, n = 3) and bone marrow (BM, n = 4) obtained from breast cancer patients. Biological validation was done in another 16 samples by RT-qPCR. Differences between CAF vs N+, CAF vs BM and N+ vs BM were represented by 20, 235 and 245 genes, respectively (SAM test, FDR < 0.01). Functional analysis revealed that genes related to development and morphogenesis were overrepresented. In a biological validation set, NOTCH2 was confirmed to be more expressed in N+ (vs CAF) and ADCY2, HECTD1, HNMT, LOX, MACF1, SLC1A3 and USP16 more expressed in BM (vs CAF). Only small differences were observed in the transcriptional profiles of fibroblasts from the primary tumor and lymph node of breast cancer patients, whereas greater differences were observed between bone marrow stromal cells and the other two sites. These differences may reflect the activities of distinct differentiation programs.

Transcriptional profile of fibroblasts obtained from the primary site, lymph node and bone marrow of breast cancer patients

PubMed Central

Del Valle, Paulo Roberto; Milani, Cintia; Brentani, Maria Mitzi; Katayama, Maria Lucia Hirata; de Lyra, Eduardo Carneiro; Carraro, Dirce Maria; Brentani, Helena; Puga, Renato; Lima, Leandro A.; Rozenchan, Patricia Bortman; Nunes, Bárbara dos Santos; Góes, João Carlos Guedes Sampaio; Azevedo Koike Folgueira, Maria Aparecida

2014-01-01

Cancer-associated fibroblasts (CAF) influence tumor development at primary as well as in metastatic sites, but there have been no direct comparisons of the transcriptional profiles of stromal cells from different tumor sites. In this study, we used customized cDNA microarrays to compare the gene expression profile of stromal cells from primary tumor (CAF, n = 4), lymph node metastasis (N+, n = 3) and bone marrow (BM, n = 4) obtained from breast cancer patients. Biological validation was done in another 16 samples by RT-qPCR. Differences between CAF vs N+, CAF vs BM and N+ vs BM were represented by 20, 235 and 245 genes, respectively (SAM test, FDR < 0.01). Functional analysis revealed that genes related to development and morphogenesis were overrepresented. In a biological validation set, NOTCH2 was confirmed to be more expressed in N+ (vs CAF) and ADCY2, HECTD1, HNMT, LOX, MACF1, SLC1A3 and USP16 more expressed in BM (vs CAF). Only small differences were observed in the transcriptional profiles of fibroblasts from the primary tumor and lymph node of breast cancer patients, whereas greater differences were observed between bone marrow stromal cells and the other two sites. These differences may reflect the activities of distinct differentiation programs. PMID:25249769

The Phenylpropanoid Pathway Is Controlled at Different Branches by a Set of R2R3-MYB C2 Repressors in Grapevine1

PubMed Central

Cavallini, Erika; Matus, José Tomás; Finezzo, Laura; Zenoni, Sara; Loyola, Rodrigo; Guzzo, Flavia; Schlechter, Rudolf; Ageorges, Agnès; Arce-Johnson, Patricio

2015-01-01

Because of the vast range of functions that phenylpropanoids possess, their synthesis requires precise spatiotemporal coordination throughout plant development and in response to the environment. The accumulation of these secondary metabolites is transcriptionally controlled by positive and negative regulators from the MYB and basic helix-loop-helix protein families. We characterized four grapevine (Vitis vinifera) R2R3-MYB proteins from the C2 repressor motif clade, all of which harbor the ethylene response factor-associated amphiphilic repression domain but differ in the presence of an additional TLLLFR repression motif found in the strong flavonoid repressor Arabidopsis (Arabidopsis thaliana) AtMYBL2. Constitutive expression of VvMYB4a and VvMYB4b in petunia (Petunia hybrida) repressed general phenylpropanoid biosynthetic genes and selectively reduced the amount of small-weight phenolic compounds. Conversely, transgenic petunia lines expressing VvMYBC2-L1 and VvMYBC2-L3 showed a severe reduction in petal anthocyanins and seed proanthocyanidins together with a higher pH of crude petal extracts. The distinct function of these regulators was further confirmed by transient expression in tobacco (Nicotiana benthamiana) leaves and grapevine plantlets. Finally, VvMYBC2-L3 was ectopically expressed in grapevine hairy roots, showing a reduction in proanthocyanidin content together with the down-regulation of structural and regulatory genes of the flavonoid pathway as revealed by a transcriptomic analysis. The physiological role of these repressors was inferred by combining the results of the functional analyses and their expression patterns in grapevine during development and in response to ultraviolet B radiation. Our results indicate that VvMYB4a and VvMYB4b may play a key role in negatively regulating the synthesis of small-weight phenolic compounds, whereas VvMYBC2-L1 and VvMYBC2-L3 may additionally fine tune flavonoid levels, balancing the inductive effects of transcriptional activators. PMID:25659381

Differential Protein Expression Profiles in Glaucomatous Trabecular Meshwork: An Evaluation Study on a Small Primary Open Angle Glaucoma Population.

PubMed

Micera, Alessandra; Quaranta, Luciano; Esposito, Graziana; Floriani, Irene; Pocobelli, Augusto; Saccà, Sergio Claudio; Riva, Ivano; Manni, Gianluca; Oddone, Francesco

2016-02-01

Primary open angle glaucoma (POAG) is a progressive optic neuropathy characterized by impaired aqueous outflow and extensive remodeling in the trabecular meshwork (TM). The aim of this study was to characterize and compare the expression patterns of selected proteins belonging to the tissue remodeling, inflammation and growth factor pathways in ex vivo glaucomatous and post-mortem TMs using protein-array analysis. TM specimens were collected from 63 white subjects, including 40 patients with glaucoma and 23 controls. Forty POAG TMs were collected at the time of surgery and 23 post-mortem specimens were from non-glaucomatous donor sclerocorneal tissues. Protein profiles were evaluated using a chip-based array consisting of 60 literature-selected antibodies. A different expression of some factors was observed in POAG TMs with respect to post-mortem specimens, either in abundance (interleukin [IL]10, IL6, IL5, IL7, IL12, IL3, macrophage inflammatory protein [MIP]1δ/α, vascular endothelial growth factor [VEGF], transforming growth factor beta 1 [TGFβ1], soluble tumor necrosis factor receptor I [sTNFRI]) or in scarcity (IL16, IL18, intercellular adhesion molecule 3 [ICAM3], matrix metalloproteinase-7 [MMP7], tissue inhibitor of metalloproteinase 1 [TIMP1]). MMP2, MMP7, TGFβ1, and VEGF expressions were confirmed by Western blot, zymography, and polymerase chain reaction. No difference in protein profile expression was detected between glaucomatous subtypes. The analysis of this small TM population highlighted some proteins linked to POAG, some previously reported and others of new detection (IL7, MIPs, sTNFαRI). A larger POAG population is required to select promising disease-associated biomarker candidates. This study was partially supported by the Fondazione Roma, the Italian Ministry of Health and the "National 5xMille 2010 tax donation to IRCCS-G.B. Bietti Foundation".

Comparison of PD-L1 mRNA Expression Measured with the CheckPoint Typer® Assay with PD-L1 Protein Expression Assessed with Immunohistochemistry in Non-small Cell Lung Cancer.

PubMed

Erber, Ramona; Stöhr, Robert; Herlein, Stefanie; Giedl, Claudia; Rieker, Ralf Joachim; Fuchs, Florian; Ficker, Joachim H; Hartmann, Arndt; Veltrup, Elke; Wirtz, Ralph M; Brueckl, Wolfgang M

2017-12-01

Immunohistochemical (IHC) assessment of programmed death-ligand 1 (PD-L1) in non-small cell lung cancer (NSCLC) has become important since the development of anti-PD-1/-PD-L1 directed drugs. Various PD-L1 antibodies and cut-offs have been used in different trials to predict response to these drugs, thus comparison of those studies is difficult. We compared PD-L1 mRNA expression measured by RT-qPCR with PD-L1 protein expression evaluated by IHC. Moreover, we investigated the impact of different tumour tissue acquisition methods on the reliability of PD-L1 measurement techniques. NSCLC cases (N=22), including n=9 mediastinal lymph node biopsies acquired by endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) and n=5 metastases, were evaluated prospectively for PD-L1 protein on tumor cells (TC) and immune cells (IC) using E1L3N and 28-8 antibodies and PD-L1 mRNA using the CheckPoint TYPER® assay. In primary NSCLC tissues, agreement between PD-L1 mRNA and TC staining using the 28-8 antibody was excellent (ĸ=0.85, p=0.0002). Comparing both PD-L1 antibodies against each other showed a kappa value of 0.58 (p=0.0106). In EBUS-TBNA, PD-L1 mRNA correlated perfectly with the 28-8 antibody (ĸ=1.0, p=0.0023). PD-L1 mRNA levels significantly differed when comparing 28-8 TC staining of tumours >49% with 1-49% and 0% (p=0.0040; p=0.0081, respectively). In metastatic lesions, differences between PD-L1 mRNA and IHC became apparent (ĸ=0.2, p=0.2525). Testing of PD-L1 mRNA and 28-8 IHC showed an excellent agreement in NSCLC samples including mediastinal lymph node biopsies. Since PD-L1 expression in >50% TC detected by 28-8 IHC can be reliably detected by RT-qPCR, quantitative PD-L1 mRNA determination should be considered as an alternative to IHC as there is no interobserver variability in RNA results. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Comprehensive Molecular Characterization of Muscle-Invasive Bladder Cancer.

PubMed

Robertson, A Gordon; Kim, Jaegil; Al-Ahmadie, Hikmat; Bellmunt, Joaquim; Guo, Guangwu; Cherniack, Andrew D; Hinoue, Toshinori; Laird, Peter W; Hoadley, Katherine A; Akbani, Rehan; Castro, Mauro A A; Gibb, Ewan A; Kanchi, Rupa S; Gordenin, Dmitry A; Shukla, Sachet A; Sanchez-Vega, Francisco; Hansel, Donna E; Czerniak, Bogdan A; Reuter, Victor E; Su, Xiaoping; de Sa Carvalho, Benilton; Chagas, Vinicius S; Mungall, Karen L; Sadeghi, Sara; Pedamallu, Chandra Sekhar; Lu, Yiling; Klimczak, Leszek J; Zhang, Jiexin; Choo, Caleb; Ojesina, Akinyemi I; Bullman, Susan; Leraas, Kristen M; Lichtenberg, Tara M; Wu, Catherine J; Schultz, Nicholaus; Getz, Gad; Meyerson, Matthew; Mills, Gordon B; McConkey, David J; Weinstein, John N; Kwiatkowski, David J; Lerner, Seth P

2017-10-19

We report a comprehensive analysis of 412 muscle-invasive bladder cancers characterized by multiple TCGA analytical platforms. Fifty-eight genes were significantly mutated, and the overall mutational load was associated with APOBEC-signature mutagenesis. Clustering by mutation signature identified a high-mutation subset with 75% 5-year survival. mRNA expression clustering refined prior clustering analyses and identified a poor-survival "neuronal" subtype in which the majority of tumors lacked small cell or neuroendocrine histology. Clustering by mRNA, long non-coding RNA (lncRNA), and miRNA expression converged to identify subsets with differential epithelial-mesenchymal transition status, carcinoma in situ scores, histologic features, and survival. Our analyses identified 5 expression subtypes that may stratify response to different treatments. Copyright © 2017 Elsevier Inc. All rights reserved.

Perturbed effects at radiation physics

NASA Astrophysics Data System (ADS)

Külahcı, Fatih; Şen, Zekâi

2013-09-01

Perturbation methodology is applied in order to assess the linear attenuation coefficient, mass attenuation coefficient and cross-section behavior with random components in the basic variables such as the radiation amounts frequently used in the radiation physics and chemistry. Additionally, layer attenuation coefficient (LAC) and perturbed LAC (PLAC) are proposed for different contact materials. Perturbation methodology provides opportunity to obtain results with random deviations from the average behavior of each variable that enters the whole mathematical expression. The basic photon intensity variation expression as the inverse exponential power law (as Beer-Lambert's law) is adopted for perturbation method exposition. Perturbed results are presented not only in terms of the mean but additionally the standard deviation and the correlation coefficients. Such perturbation expressions provide one to assess small random variability in basic variables.

Regulation of bacterial photosynthesis genes by the small noncoding RNA PcrZ

PubMed Central

Mank, Nils N.; Berghoff, Bork A.; Hermanns, Yannick N.; Klug, Gabriele

2012-01-01

The small RNA PcrZ (photosynthesis control RNA Z) of the facultative phototrophic bacterium Rhodobacter sphaeroides is induced upon a drop of oxygen tension with similar kinetics to those of genes for components of photosynthetic complexes. High expression of PcrZ depends on PrrA, the response regulator of the PrrB/PrrA two-component system with a central role in redox regulation in R. sphaeroides. In addition the FnrL protein, an activator of some photosynthesis genes at low oxygen tension, is involved in redox-dependent expression of this small (s)RNA. Overexpression of full-length PcrZ in R. sphaeroides affects expression of a small subset of genes, most of them with a function in photosynthesis. Some mRNAs from the photosynthetic gene cluster were predicted to be putative PcrZ targets and results from an in vivo reporter system support these predictions. Our data reveal a negative effect of PcrZ on expression of its target mRNAs. Thus, PcrZ counteracts the redox-dependent induction of photosynthesis genes, which is mediated by protein regulators. Because PrrA directly activates photosynthesis genes and at the same time PcrZ, which negatively affects photosynthesis gene expression, this is one of the rare cases of an incoherent feed-forward loop including an sRNA. Our data identified PcrZ as a trans acting sRNA with a direct regulatory function in formation of photosynthetic complexes and provide a model for the control of photosynthesis gene expression by a regulatory network consisting of proteins and a small noncoding RNA. PMID:22988125

Regulation of bacterial photosynthesis genes by the small noncoding RNA PcrZ.

PubMed

Mank, Nils N; Berghoff, Bork A; Hermanns, Yannick N; Klug, Gabriele

2012-10-02

The small RNA PcrZ (photosynthesis control RNA Z) of the facultative phototrophic bacterium Rhodobacter sphaeroides is induced upon a drop of oxygen tension with similar kinetics to those of genes for components of photosynthetic complexes. High expression of PcrZ depends on PrrA, the response regulator of the PrrB/PrrA two-component system with a central role in redox regulation in R. sphaeroides. In addition the FnrL protein, an activator of some photosynthesis genes at low oxygen tension, is involved in redox-dependent expression of this small (s)RNA. Overexpression of full-length PcrZ in R. sphaeroides affects expression of a small subset of genes, most of them with a function in photosynthesis. Some mRNAs from the photosynthetic gene cluster were predicted to be putative PcrZ targets and results from an in vivo reporter system support these predictions. Our data reveal a negative effect of PcrZ on expression of its target mRNAs. Thus, PcrZ counteracts the redox-dependent induction of photosynthesis genes, which is mediated by protein regulators. Because PrrA directly activates photosynthesis genes and at the same time PcrZ, which negatively affects photosynthesis gene expression, this is one of the rare cases of an incoherent feed-forward loop including an sRNA. Our data identified PcrZ as a trans acting sRNA with a direct regulatory function in formation of photosynthetic complexes and provide a model for the control of photosynthesis gene expression by a regulatory network consisting of proteins and a small noncoding RNA.

Identification of transcribed sequences in Arabidopsis thaliana by using high-resolution genome tiling arrays

PubMed Central

Stolc, Viktor; Samanta, Manoj Pratim; Tongprasit, Waraporn; Sethi, Himanshu; Liang, Shoudan; Nelson, David C.; Hegeman, Adrian; Nelson, Clark; Rancour, David; Bednarek, Sebastian; Ulrich, Eldon L.; Zhao, Qin; Wrobel, Russell L.; Newman, Craig S.; Fox, Brian G.; Phillips, George N.; Markley, John L.; Sussman, Michael R.

2005-01-01

Using a maskless photolithography method, we produced DNA oligonucleotide microarrays with probe sequences tiled throughout the genome of the plant Arabidopsis thaliana. RNA expression was determined for the complete nuclear, mitochondrial, and chloroplast genomes by tiling 5 million 36-mer probes. These probes were hybridized to labeled mRNA isolated from liquid grown T87 cells, an undifferentiated Arabidopsis cell culture line. Transcripts were detected from at least 60% of the nearly 26,330 annotated genes, which included 151 predicted genes that were not identified previously by a similar genome-wide hybridization study on four different cell lines. In comparison with previously published results with 25-mer tiling arrays produced by chromium masking-based photolithography technique, 36-mer oligonucleotide probes were found to be more useful in identifying intron–exon boundaries. Using two-dimensional HPLC tandem mass spectrometry, a small-scale proteomic analysis was performed with the same cells. A large amount of strongly hybridizing RNA was found in regions “antisense” to known genes. Similarity of antisense activities between the 25-mer and 36-mer data sets suggests that it is a reproducible and inherent property of the experiments. Transcription activities were also detected for many of the intergenic regions and the small RNAs, including tRNA, small nuclear RNA, small nucleolar RNA, and microRNA. Expression of tRNAs correlates with genome-wide amino acid usage. PMID:15755812

Using an AMACR (P504S)/34betaE12/p63 cocktail for the detection of small focal prostate carcinoma in needle biopsy specimens.

PubMed

Jiang, Zhong; Li, Cuizhen; Fischer, Andrew; Dresser, Karen; Woda, Bruce A

2005-02-01

We assessed the usefulness of immunohistochemical analysis with a 3-antibody cocktail (alpha-methylacyl coenzyme A racemase [AMACR, or P504S], 34betaE12, p63) and a double-chromogen reaction for detection of limited prostate cancer in 138 needle biopsy specimens, including 82 with small foci of prostatic adenocarcinoma and 56 benign prostates. When carcinoma was present, red cytoplasmic granular staining (AMACR) in the malignant glands and cells and dark brown nuclear (p63) and cytoplasmic (34betaE12) staining in basal cells of adjacent nonmalignant glands were found. Of 82 cases of small foci of prostatic adenocarcinoma, 78 (95%) expressed AMACR; all malignant glands were negative for basal cell staining. All benign glands adjacent to malignant glands were recognized easily by basal cell marker positivity and little or no AMACR expression. No benign glands were simultaneously positive for AMACR and negative for basal cell markers (specificity, 100%). There were no differences in intensity and numbers of positive glands with double-chromogen staining compared with using 1-color staining. Our results indicate that immunohistochemistry with a 3-antibody cocktail and double chromogen is a simple and easy assay that can be used as a routine test, which overcomes the problems of studying small lesions in prostate needle biopsies with multiple immunohistochemical stains.

Identification of transcribed sequences in Arabidopsis thaliana by using high-resolution genome tiling arrays

NASA Technical Reports Server (NTRS)

Stolc, Viktor; Samanta, Manoj Pratim; Tongprasit, Waraporn; Sethi, Himanshu; Liang, Shoudan; Nelson, David C.; Hegeman, Adrian; Nelson, Clark; Rancour, David; Bednarek, Sebastian;

[What is the prognostic significance of histomorphology in small cell lung carcinoma?].

PubMed

Facilone, F; Cimmino, A; Assennato, G; Sardelli, P; Colucci, G A; Resta, L

1993-01-01

What is the prognostic significant of the histomorphology in the small cell carcinomas of the lung? After the WHO classification of the lung cancer (1981), several studies criticized the subdivision of the small cell carcinoma in three sub-types (oat-cell, intermediate cell and combined types). The role of histology in the prognostic predition has been devaluated. In order to verify the prognostic value of the morphology of the small cell types of lung cancer, we performed a multivariate analysis in 62 patients. The survival rate was analytically compared with the following parameters: nuclear maximum diameter, nuclear form, nuclear chromatism, chromatine distribution, presence of nucleolus, evidence of cytoplasm. The results showed that none of these parameters are able to express a prognostic value. According to the recent studies, we think that the small cell carcinoma of the lung is a neoplasia with a multiform histologic pattern. Differences observed in clinical management are not correlate with the morphology, but with other biological parameters still unknown.

Gene expression in gastrointestinal stromal tumors is distinguished by KIT genotype and anatomic site.

PubMed

Antonescu, Cristina R; Viale, Agnes; Sarran, Lisa; Tschernyavsky, Sylvia J; Gonen, Mithat; Segal, Neil H; Maki, Robert G; Socci, Nicholas D; DeMatteo, Ronald P; Besmer, Peter

2004-05-15

Gastrointestinal stromal tumors (GISTs) are specific KIT expressing and KIT-signaling driven mesenchymal tumors of the human digestive tract, many of which have KIT-activating mutations. Previous studies have found a relatively homogeneous gene expression profile in GIST, as compared with other histological types of sarcomas. Transcriptional heterogeneity within clinically or molecularly defined subsets of GISTs has not been previously reported. We tested the hypothesis that the gene expression profile in GISTs might be related to KIT genotype and possibly to other clinicopathological factors. An HG-U133A Affymetrix chip (22,000 genes) platform was used to determine the variability of gene expression in 28 KIT-expressing GIST samples from 24 patients. A control group of six intra-abdominal leiomyosarcomas was also included for comparison. Statistical analyses (t tests) were performed to identify discriminatory gene lists among various GIST subgroups. The levels of expression of various GIST subsets were also linked to a modified version of the growth factor/KIT signaling pathway to analyze differences at various steps in signal transduction. Genes involved in KIT signaling were differentially expressed among wild-type and mutant GISTs. High gene expression of potential drug targets, such as VEGF, MCSF, and BCL2 in the wild-type group, and Mesothelin in exon 9 GISTs were found. There was a striking difference in gene expression between stomach and small bowel GISTs. This finding was validated in four separate tumors, two gastric and two intestinal, from a patient with familial GIST with a germ-line KIT W557R substitution. GISTs have heterogeneous gene expression depending on KIT genotype and tumor location, which is seen at both the genomic level and the KIT signaling pathway in particular. These findings may explain their variable clinical behavior and response to therapy.

Postnatal overfeeding promotes early onset and exaggeration of high-fat diet-induced nonalcoholic fatty liver disease through disordered hepatic lipid metabolism in rats.

PubMed

Ji, Chenlin; Dai, Yanyan; Jiang, Weiwei; Liu, Juan; Hou, Miao; Wang, Junle; Burén, Jonas; Li, Xiaonan

2014-11-01

Exposure to overnutrition in critical or sensitive developmental periods may increase the risk of developing obesity and metabolic syndrome in adults. Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome, but the relationship among postnatal nutrition, lipid metabolism, and NAFLD progression during development remains poorly understood. Here we investigated in a rat model whether postnatal overfeeding increases susceptibility to NAFLD in response to a high-fat diet. Litters from Sprague-Dawley dams were culled to three (small litters) or ten (normal litters) pups and then weaned onto a standard or high-fat diet at postnatal day 21 to generate normal-litter, small-litter, normal-litter/high-fat, and small-litter/high-fat groups. At age 16 weeks, the small-litter and both high-fat groups showed obesity, dyslipidemia, and insulin resistance. Hepatic disorders appeared earlier in the small-litter/high-fat rats with greater liver mass gain and higher hepatic triglycerides and steatosis score versus normal-litter/high-fat rats. Hepatic acetyl-CoA carboxylase activity and mRNA expression were increased in small-litter rats and aggravated in small-litter/high-fat rats but not in normal-litter/high-fat rats. The high expression in small-litter/high-fat rats coincided with high sterol regulatory element-binding protein-1c mRNA and protein expression. However, mRNA expression of enzymes involved in hepatic fatty acid oxidation (carnitine palmitoyltransferase 1) and output (microsomal triglyceride transfer protein) was decreased under a high-fat diet regardless of litter size. In conclusion, overfeeding related to small-litter rearing during lactation contributes to the NAFLD phenotype when combined with a high-fat diet, possibly through up-regulated hepatic lipogenesis. Copyright © 2014 Elsevier Inc. All rights reserved.

MicroRNA-429 induces tumorigenesis of human non-small cell lung cancer cells and targets multiple tumor suppressor genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lang, Yaoguo; Xu, Shidong; Ma, Jianqun

2014-07-18

Highlights: • MiR-429 expression is upregulated in non-small cell lung cancer (NSCLC). • MiR-429 inhibits PTEN, RASSF8 and TIMP2 expression. • MiR-429 promotes metastasis and proliferation. • We report important regulatory mechanisms involved in NSCLC progression. • MiR-429 is a potential therapeutic target and diagnostic marker. - Abstract: Lung cancer is the major cause of cancer death globally. MicroRNAs are evolutionally conserved small noncoding RNAs that are critical for the regulation of gene expression. Aberrant expression of microRNA (miRNA) has been implicated in cancer initiation and progression. In this study, we demonstrated that the expression of miR-429 are often upregulatedmore » in non-small cell lung cancer (NSCLC) compared with normal lung tissues, and its expression level is also increased in NSCLC cell lines compared with normal lung cells. Overexpression of miR-429 in A549 NSCLC cells significantly promoted cell proliferation, migration and invasion, whereas inhibition of miR-429 inhibits these effects. Furthermore, we demonstrated that miR-429 down-regulates PTEN, RASSF8 and TIMP2 expression by directly targeting the 3′-untranslated region of these target genes. Taken together, our results suggest that miR-429 plays an important role in promoting the proliferation and metastasis of NSCLC cells and is a potential target for NSCLC therapy.« less

Simultaneous sequencing of coding and noncoding RNA reveals a human transcriptome dominated by a small number of highly expressed noncoding genes.

PubMed

Boivin, Vincent; Deschamps-Francoeur, Gabrielle; Couture, Sonia; Nottingham, Ryan M; Bouchard-Bourelle, Philia; Lambowitz, Alan M; Scott, Michelle S; Abou-Elela, Sherif

2018-07-01

Comparing the abundance of one RNA molecule to another is crucial for understanding cellular functions but most sequencing techniques can target only specific subsets of RNA. In this study, we used a new fragmented ribodepleted TGIRT sequencing method that uses a thermostable group II intron reverse transcriptase (TGIRT) to generate a portrait of the human transcriptome depicting the quantitative relationship of all classes of nonribosomal RNA longer than 60 nt. Comparison between different sequencing methods indicated that FRT is more accurate in ranking both mRNA and noncoding RNA than viral reverse transcriptase-based sequencing methods, even those that specifically target these species. Measurements of RNA abundance in different cell lines using this method correlate with biochemical estimates, confirming tRNA as the most abundant nonribosomal RNA biotype. However, the single most abundant transcript is 7SL RNA, a component of the signal recognition particle. S tructured n on c oding RNAs (sncRNAs) associated with the same biological process are expressed at similar levels, with the exception of RNAs with multiple functions like U1 snRNA. In general, sncRNAs forming RNPs are hundreds to thousands of times more abundant than their mRNA counterparts. Surprisingly, only 50 sncRNA genes produce half of the non-rRNA transcripts detected in two different cell lines. Together the results indicate that the human transcriptome is dominated by a small number of highly expressed sncRNAs specializing in functions related to translation and splicing. © 2018 Boivin et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Association between ethnicity and obesity with high-density lipoprotein (HDL) function and subclass distribution.

PubMed

Woudberg, Nicholas J; Goedecke, Julia H; Blackhurst, Dee; Frias, Miguel; James, Richard; Opie, Lionel H; Lecour, Sandrine

2016-05-11

Obesity and low high-density lipoprotein-cholesterol (HDL-C) levels are associated with cardiovascular risk. Surprisingly, despite a greater prevalence of obesity and lower HDL concentrations than white women, black South African women are relatively protected against ischaemic heart disease. We investigated whether this apparent discrepancy may be related to different HDL function and subclass distribution in black and white, normal-weight and obese South African women (n = 40). HDL functionality was assessed by measuring paraoxonase (PON) activity, platelet activating factor acetylhydrolase (PAF-AH) activity, Oxygen Radical Absorbance Capacity (ORAC) and quantification of the expression of vascular cell adhesion molecule in endothelial cells. PON-1 and PAF-AH expression was determined in isolated HDL and serum using Western blotting. Levels of large, intermediate and small HDL subclasses were measured using the Lipoprint® system. PON activity was lower in white compared to black women (0.49 ± 0.09 U/L vs 0.78 ± 0.10 U/L, p < 0.05), regardless of PON-1 protein levels. Obese black women had lower PAF-AH activity (9.34 ± 1.15 U/L vs 13.89 ± 1.21 U/L, p <0.05) and HDL-associated PAF-AH expression compared to obese white women. Compared to normal-weight women, obese women had lower large HDL, greater intermediate and small HDL; an effect that was more pronounced in white women than black women. There were no differences in antioxidant capacity or anti-inflammatory function across groups. Our data show that both obesity and ethnicity are associated with differences in HDL functionality, while obesity was associated with decreases in large HDL subclass distribution. Measuring HDL functionality and subclass may, therefore, be important factors to consider when assessing cardiovascular risk.

[Evaluation of three-dimensional tumor microvascular architecture phenotype heterogeneity in non-small cell carcinoma and its significance].

PubMed

Zhou, Hui; Liu, Jinkang; Chen, Shengxi; Xiong, Zeng; Zhou, Jianhua; Tong, Shiyu; Chen, Hao; Zhou, Moling

2012-06-01

To explore the degree, mechanism and clinical significance of three-dimensional tumor microvascular architecture phenotype heterogeneity (3D-TMAPH) in non-small cell carcinoma (NSCLC). Twenty-one samples of solitary pulmonary nodules were collected integrally. To establish two-dimensional tumor microvascular architecture phenotype (2D-TMAP) and three-dimensional tumor microvascular architecture phenotype (3D-TMAP), five layers of each nodule were selected and embedded in paraffin. Test indices included the expressions of vascular endothelial growth factor (VEGF), proliferating cell nuclear antigen (PCNA), EphB4, ephfinB2 and microvascular density marked by anti-CD34 (CD34-MVD). The degrees of 3D-TMAPH were evaluated by the coefficient of variation and extend of heterogeneity. Spearman rank correlation analysis was used to investigate the relationships between 2D-TMAP, 3D-TMAP and clinicopathological features. 3D-TMAPH showed that 2D-TMAP heterogeneity was expressed in the tissues of NSCLC. The heterogeneities in the malignant nodules were significantly higher than those in the active inflammatory nodules and tubercular nodules. In addition, different degrees of heterogeneity of CD34-MVD and PCNA were found in NSCLC tissues. The coefficients of variation of CD34- MVD and PCNA were positively related to the degree of differentiation (all P<0.05), but not related to the P-TNM stages, histological type or lymphatic metastasis (all P>0.05). The level of heterogeneity of various expression indexes (ephrinB2, EphB4, VEGF) in NSCLC tissues were inconsistent, but there were no significant differences in heterogeneity in NSCLC tissues with different histological types (P>0.05). 3D-TMAPH exists widely in the microenvironment during the genesis and development of NSCLC and has a significant impact on its biological complexity.

Examination of the genetic basis for sexual dimorphism in the Aedes aegypti (dengue vector mosquito) pupal brain

PubMed Central

2014-01-01

Background Most animal species exhibit sexually dimorphic behaviors, many of which are linked to reproduction. A number of these behaviors, including blood feeding in female mosquitoes, contribute to the global spread of vector-borne illnesses. However, knowledge concerning the genetic basis of sexually dimorphic traits is limited in any organism, including mosquitoes, especially with respect to differences in the developing nervous system. Methods Custom microarrays were used to examine global differences in female vs. male gene expression in the developing pupal head of the dengue vector mosquito, Aedes aegypti. The spatial expression patterns of a subset of differentially expressed transcripts were examined in the developing female vs. male pupal brain through in situ hybridization experiments. Small interfering RNA (siRNA)-mediated knockdown studies were used to assess the putative role of Doublesex, a terminal component of the sex determination pathway, in the regulation of sex-specific gene expression observed in the developing pupal brain. Results Transcripts (2,527), many of which were linked to proteolysis, the proteasome, metabolism, catabolic, and biosynthetic processes, ion transport, cell growth, and proliferation, were found to be differentially expressed in A. aegypti female vs. male pupal heads. Analysis of the spatial expression patterns for a subset of dimorphically expressed genes in the pupal brain validated the data set and also facilitated the identification of brain regions with dimorphic gene expression. In many cases, dimorphic gene expression localized to the optic lobe. Sex-specific differences in gene expression were also detected in the antennal lobe and mushroom body. siRNA-mediated gene targeting experiments demonstrated that Doublesex, a transcription factor with consensus binding sites located adjacent to many dimorphically expressed transcripts that function in neural development, is required for regulation of sex-specific gene expression in the developing A. aegypti brain. Conclusions These studies revealed sex-specific gene expression profiles in the developing A. aegypti pupal head and identified Doublesex as a key regulator of sexually dimorphic gene expression during mosquito neural development. PMID:25729562

Protein composition correlates with the mechanical properties of spider ( Argiope trifasciata ) dragline silk.

PubMed

Marhabaie, Mohammad; Leeper, Thomas C; Blackledge, Todd A

2014-01-13

We investigated the natural variation in silk composition and mechanical performance of the orb-weaving spider Argiope trifasciata at multiple spatial and temporal scales in order to assess how protein composition contributes to the remarkable material properties of spider dragline silk. Major ampullate silk in orb-weaving spiders consists predominantly of two proteins (MaSp1 and MaSp2) with divergent amino acid compositions and functionally different microstructures. Adjusting the expression of these two proteins therefore provides spiders with a simple mechanism to alter the material properties of their silk. We first assessed the reliability and precision of the Waters AccQ-Tag amino acid composition analysis kit for determining the amino acid composition of small quantities of spider silk. We then tested how protein composition varied within single draglines, across draglines spun by the same spider on different days, and finally between spiders. Then, we correlated chemical composition with the material properties of dragline silk. Overall, we found that the chemical composition of major ampullate silk was in general homogeneous among individuals of the same population. Variation in chemical composition was not detectable within silk spun by a single spider on a single day. However, we found that variation within a single spider's silk across different days could, in rare instances, be greater than variation among individual spiders. Most of the variation in silk composition in our investigation resulted from a small number of outliers (three out of sixteen individuals) with a recent history of stress, suggesting stress affects silk production process in orb web spiders. Based on reported sequences for MaSp genes, we developed a gene expression model showing the covariation of the most abundant amino acids in major ampullate silk. Our gene expression model supports that dragline silk composition was mostly determined by the relative abundance of MaSp1 and MaSp2. Finally, we showed that silk composition (especially proline content) strongly correlated with some measures of mechanical performance, particularly how much fibers shrunk during supercontraction as well as their breaking strains. Our findings suggest that spiders are able to change the relative expression rates of different MaSp genes to produce silk fibers with different chemical compositions, and hence, different material properties.

Expression profiling of snoRNAs in normal hematopoiesis and AML

PubMed Central

Warner, Wayne A.; Spencer, David H.; Trissal, Maria; White, Brian S.; Helton, Nichole; Ley, Timothy J.

2018-01-01

Small nucleolar RNAs (snoRNAs) are noncoding RNAs that contribute to ribosome biogenesis and RNA splicing by modifying ribosomal RNA and spliceosome RNAs, respectively. We optimized a next-generation sequencing approach and a custom analysis pipeline to identify and quantify expression of snoRNAs in acute myeloid leukemia (AML) and normal hematopoietic cell populations. We show that snoRNAs are expressed in a lineage- and development-specific fashion during hematopoiesis. The most striking examples involve snoRNAs located in 2 imprinted loci, which are highly expressed in hematopoietic progenitors and downregulated during myeloid differentiation. Although most snoRNAs are expressed at similar levels in AML cells compared with CD34+, a subset of snoRNAs showed consistent differential expression, with the great majority of these being decreased in the AML samples. Analysis of host gene expression, splicing patterns, and whole-genome sequence data for mutational events did not identify transcriptional patterns or genetic alterations that account for these expression differences. These data provide a comprehensive analysis of the snoRNA transcriptome in normal and leukemic cells and should be helpful in the design of studies to define the contribution of snoRNAs to normal and malignant hematopoiesis. PMID:29365324

Diagnostic implications of IDH1-R132H and OLIG2 expression patterns in rare and challenging glioblastoma variants.

PubMed

Joseph, Nancy M; Phillips, Joanna; Dahiya, Sonika; M Felicella, Michelle; Tihan, Tarik; Brat, Daniel J; Perry, Arie

2013-03-01

Recent work has demonstrated that nearly all diffuse gliomas display nuclear immunoreactivity for the bHLH transcription factor OLIG2, and the R132H mutant isocitrate dehydrogenase 1 (IDH1) protein is expressed in the majority of diffuse gliomas other than primary glioblastoma. However, these antibodies have not been widely applied to rarer glioblastoma variants, which can be diagnostically challenging when the astrocytic features are subtle. We therefore surveyed the expression patterns of OLIG2 and IDH1 in 167 non-conventional glioblastomas, including 45 small cell glioblastomas, 45 gliosarcomas, 34 glioblastomas with primitive neuroectodermal tumor-like foci (PNET-like foci), 23 with an oligodendroglial component, 11 granular cell glioblastomas, and 9 giant cell glioblastomas. OLIG2 was strongly expressed in all glioblastomas with oligodendroglial component, 98% of small cell glioblastomas, and all granular cell glioblastomas, the latter being particularly helpful in ruling out macrophage-rich lesions. In 74% of glioblastomas with PNET-like foci, OLIG2 expression was retained in the PNET-like foci, providing a useful distinction from central nervous system PNETs. The glial component of gliosarcomas was OLIG2 positive in 93% of cases, but only 14% retained focal expression in the sarcomatous component; as such this marker would not reliably distinguish these from pure sarcoma in most cases. OLIG2 was expressed in 67% of giant cell glioblastomas. IDH1 was expressed in 55% of glioblastomas with oligodendroglial component, 15% of glioblastomas with PNET-like foci, 7% of gliosarcomas, and none of the small cell, granular cell, or giant cell glioblastomas. This provides further support for the notion that most glioblastomas with oligodendroglial component are secondary, while small cell glioblastomas, granular cell glioblastomas, and giant cell glioblastomas are primary variants. Therefore, in one of the most challenging differential diagnoses, IDH1 positivity could provide strong support for glioblastoma with oligodendroglial component, while essentially excluding small cell glioblastoma.

SEGEL: A Web Server for Visualization of Smoking Effects on Human Lung Gene Expression.

PubMed

Xu, Yan; Hu, Brian; Alnajm, Sammy S; Lu, Yin; Huang, Yangxin; Allen-Gipson, Diane; Cheng, Feng

2015-01-01

Cigarette smoking is a major cause of death worldwide resulting in over six million deaths per year. Cigarette smoke contains complex mixtures of chemicals that are harmful to nearly all organs of the human body, especially the lungs. Cigarette smoking is considered the major risk factor for many lung diseases, particularly chronic obstructive pulmonary diseases (COPD) and lung cancer. However, the underlying molecular mechanisms of smoking-induced lung injury associated with these lung diseases still remain largely unknown. Expression microarray techniques have been widely applied to detect the effects of smoking on gene expression in different human cells in the lungs. These projects have provided a lot of useful information for researchers to understand the potential molecular mechanism(s) of smoke-induced pathogenesis. However, a user-friendly web server that would allow scientists to fast query these data sets and compare the smoking effects on gene expression across different cells had not yet been established. For that reason, we have integrated eight public expression microarray data sets from trachea epithelial cells, large airway epithelial cells, small airway epithelial cells, and alveolar macrophage into an online web server called SEGEL (Smoking Effects on Gene Expression of Lung). Users can query gene expression patterns across these cells from smokers and nonsmokers by gene symbols, and find the effects of smoking on the gene expression of lungs from this web server. Sex difference in response to smoking is also shown. The relationship between the gene expression and cigarette smoking consumption were calculated and are shown in the server. The current version of SEGEL web server contains 42,400 annotated gene probe sets represented on the Affymetrix Human Genome U133 Plus 2.0 platform. SEGEL will be an invaluable resource for researchers interested in the effects of smoking on gene expression in the lungs. The server also provides useful information for drug development against smoking-related diseases. The SEGEL web server is available online at http://www.chengfeng.info/smoking_database.html.

Inhibition of small-intestinal sugar absorption mediated by sodium orthovanadate Na3VO4 in rats and its mechanisms

PubMed Central

Ai, Jing; Du, Jie; Wang, Ning; Du, Zhi-Min; Yang, Bao-Feng

2004-01-01

AIM: To investigate the inhibitory effects of sodium orthovanadate on small-intestinal glucose and maltose absorption in rats and its mechanism. METHODS: Normal Wistar rats were lavaged with sodium orthovanadate (16 mg/kg, 4 mg/kg and 1 mg/kg) for 6 d. Blood glucose values were measured after fasting and 0.5, 1, 1.5 and 2 h after glucose and maltose feeding with oxidation-enzyme method. α-glucosidase was abstracted from the upper small intestine, and its activity was examined. mRNA expression of α-glucosidase and glucose-transporter 2 (GLUT2) in epithelial cells of the small intestine was observed by in situ hybridization. RESULTS: Sodium orthovanadate could delay the increase of plasma glucose concentration after glucose and maltose loading, area under curve (AUC) in these groups was lower than that in control group. Sodium orthovanadate at dosages of 10 μmol/L, 100 μmol/L and 1000 μmol/L could suppress the activity of α-glucosidase in the small intestine of normal rats, with an inhibition rate of 68.18%, 87.22% and 91.91%, respectively. Sodium orthovanadate reduced mRNA expression of α-glucosidase and GLUT2 in epithelial cells of small intestine. CONCLUSION: Sodium orthovanadate can reduce and delay the absorption of glucose and maltose. The mechanism may be that it can inhibit the activity and mRNA expression of α-glucosidase, as well as mRNA expression of GLUT2 in small intestine. PMID:15534916

Global Analysis of Small RNA Dynamics during Seed Development of Picea glauca and Arabidopsis thaliana Populations Reveals Insights on their Evolutionary Trajectories

PubMed Central

Liu, Yang; El-Kassaby, Yousry A.

2017-01-01

While DNA methylation carries genetic signals and is instrumental in the evolution of organismal complexity, small RNAs (sRNAs), ~18–24 ribonucleotide (nt) sequences, are crucial mediators of methylation as well as gene silencing. However, scant study deals with sRNA evolution via featuring their expression dynamics coupled with species of different evolutionary time. Here we report an atlas of sRNAs and microRNAs (miRNAs, single-stranded sRNAs) produced over time at seed-set of two major spermatophytes represented by populations of Picea glauca and Arabidopsis thaliana with different seed-set duration. We applied diverse profiling methods to examine sRNA and miRNA features, including size distribution, sequence conservation and reproduction-specific regulation, as well as to predict their putative targets. The top 27 most abundant miRNAs were highly overlapped between the two species (e.g., miR166,−319 and−396), but in P. glauca, they were less abundant and significantly less correlated with seed-set phases. The most abundant sRNAs in libraries were deeply conserved miRNAs in the plant kingdom for Arabidopsis but long sRNAs (24-nt) for P. glauca. We also found significant difference in normalized expression between populations for population-specific sRNAs but not for lineage-specific ones. Moreover, lineage-specific sRNAs were enriched in the 21-nt size class. This pattern is consistent in both species and alludes to a specific type of sRNAs (e.g., miRNA, tasiRNA) being selected for. In addition, we deemed 24 and 9 sRNAs in P. glauca and Arabidopsis, respectively, as sRNA candidates targeting known adaptive genes. Temperature had significant influence on selected gene and miRNA expression at seed development in both species. This study increases our integrated understanding of sRNA evolution and its potential link to genomic architecture (e.g., sRNA derivation from genome and sRNA-mediated genomic events) and organismal complexity (e.g., association between different sRNA expression and their functionality). PMID:29046688

A Leveraged Signal-to-Noise Ratio (LSTNR) Method to Extract Differentially Expressed Genes and Multivariate Patterns of Expression From Noisy and Low-Replication RNAseq Data

PubMed Central

Lozoya, Oswaldo A.; Santos, Janine H.; Woychik, Richard P.

2018-01-01

To life scientists, one important feature offered by RNAseq, a next-generation sequencing tool used to estimate changes in gene expression levels, lies in its unprecedented resolution. It can score countable differences in transcript numbers among thousands of genes and between experimental groups, all at once. However, its high cost limits experimental designs to very small sample sizes, usually N = 3, which often results in statistically underpowered analysis and poor reproducibility. All these issues are compounded by the presence of experimental noise, which is harder to distinguish from instrumental error when sample sizes are limiting (e.g., small-budget pilot tests), experimental populations exhibit biologically heterogeneous or diffuse expression phenotypes (e.g., patient samples), or when discriminating among transcriptional signatures of closely related experimental conditions (e.g., toxicological modes of action, or MOAs). Here, we present a leveraged signal-to-noise ratio (LSTNR) thresholding method, founded on generalized linear modeling (GLM) of aligned read detection limits to extract differentially expressed genes (DEGs) from noisy low-replication RNAseq data. The LSTNR method uses an agnostic independent filtering strategy to define the dynamic range of detected aggregate read counts per gene, and assigns statistical weights that prioritize genes with better sequencing resolution in differential expression analyses. To assess its performance, we implemented the LSTNR method to analyze three separate datasets: first, using a systematically noisy in silico dataset, we demonstrated that LSTNR can extract pre-designed patterns of expression and discriminate between “noise” and “true” differentially expressed pseudogenes at a 100% success rate; then, we illustrated how the LSTNR method can assign patient-derived breast cancer specimens correctly to one out of their four reported molecular subtypes (luminal A, luminal B, Her2-enriched and basal-like); and last, we showed the ability to retrieve five different modes of action (MOA) elicited in livers of rats exposed to three toxicants under three nutritional routes by using the LSTNR method. By combining differential measurements with resolving power to detect DEGs, the LSTNR method offers an alternative approach to interrogate noisy and low-replication RNAseq datasets, which handles multiple biological conditions at once, and defines benchmarks to validate RNAseq experiments with standard benchtop assays. PMID:29868123

Differences and similarities between human and chimpanzee neural progenitors during cerebral cortex development

PubMed Central

Mora-Bermúdez, Felipe; Badsha, Farhath; Kanton, Sabina; Camp, J Gray; Vernot, Benjamin; Köhler, Kathrin; Voigt, Birger; Okita, Keisuke; Maricic, Tomislav; He, Zhisong; Lachmann, Robert; Pääbo, Svante; Treutlein, Barbara; Huttner, Wieland B

2016-01-01

Human neocortex expansion likely contributed to the remarkable cognitive abilities of humans. This expansion is thought to primarily reflect differences in proliferation versus differentiation of neural progenitors during cortical development. Here, we have searched for such differences by analysing cerebral organoids from human and chimpanzees using immunohistofluorescence, live imaging, and single-cell transcriptomics. We find that the cytoarchitecture, cell type composition, and neurogenic gene expression programs of humans and chimpanzees are remarkably similar. Notably, however, live imaging of apical progenitor mitosis uncovered a lengthening of prometaphase-metaphase in humans compared to chimpanzees that is specific to proliferating progenitors and not observed in non-neural cells. Consistent with this, the small set of genes more highly expressed in human apical progenitors points to increased proliferative capacity, and the proportion of neurogenic basal progenitors is lower in humans. These subtle differences in cortical progenitors between humans and chimpanzees may have consequences for human neocortex evolution. DOI: http://dx.doi.org/10.7554/eLife.18683.001 PMID:27669147

The quantitative proteomes of human-induced pluripotent stem cells and embryonic stem cells

PubMed Central

Munoz, Javier; Low, Teck Y; Kok, Yee J; Chin, Angela; Frese, Christian K; Ding, Vanessa; Choo, Andre; Heck, Albert J R

2011-01-01

Assessing relevant molecular differences between human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) is important, given that such differences may impact their potential therapeutic use. Controversy surrounds recent gene expression studies comparing hiPSCs and hESCs. Here, we present an in-depth quantitative mass spectrometry-based analysis of hESCs, two different hiPSCs and their precursor fibroblast cell lines. Our comparisons confirmed the high similarity of hESCs and hiPSCS at the proteome level as 97.8% of the proteins were found unchanged. Nevertheless, a small group of 58 proteins, mainly related to metabolism, antigen processing and cell adhesion, was found significantly differentially expressed between hiPSCs and hESCs. A comparison of the regulated proteins with previously published transcriptomic studies showed a low overlap, highlighting the emerging notion that differences between both pluripotent cell lines rather reflect experimental conditions than a recurrent molecular signature. PMID:22108792

Study on characteristics of the aperture-averaging factor of atmospheric scintillation in terrestrial optical wireless communication

NASA Astrophysics Data System (ADS)

Shen, Hong; Liu, Wen-xing; Zhou, Xue-yun; Zhou, Li-ling; Yu, Long-Kun

2018-02-01

In order to thoroughly understand the characteristics of the aperture-averaging effect of atmospheric scintillation in terrestrial optical wireless communication and provide references for engineering design and performance evaluation of the optics system employed in the atmosphere, we have theoretically deduced the generally analytic expression of the aperture-averaging factor of atmospheric scintillation, and numerically investigated characteristics of the apertureaveraging factor under different propagation conditions. The limitations of the current commonly used approximate calculation formula of aperture-averaging factor have been discussed, and the results showed that the current calculation formula is not applicable for the small receiving aperture under non-uniform turbulence link. Numerical calculation has showed that aperture-averaging factor of atmospheric scintillation presented an exponential decline model for the small receiving aperture under non-uniform turbulent link, and the general expression of the model was given. This model has certain guiding significance for evaluating the aperture-averaging effect in the terrestrial optical wireless communication.

Exploring miRNA based approaches in cancer diagnostics and therapeutics.

PubMed

Mishra, Shivangi; Yadav, Tanuja; Rani, Vibha

2016-02-01

MicroRNAs (miRNAs), a highly conserved class of tissue specific, small non-protein coding RNAs maintain cell homeostasis by negative gene regulation. Proper controlling of miRNA expression is required for a balanced physiological environment, as these small molecules influence almost every genetic pathway from cell cycle checkpoint, cell proliferation to apoptosis, with a wide range of target genes. Deregulation in miRNAs expression correlates with various cancers by acting as tumor suppressors and oncogenes. Although promising therapies exist to control tumor development and progression, there is a lack of efficient diagnostic and therapeutic approaches for delineating various types of cancer. The molecularly different tumors can be differentiated by specific miRNA profiling as their phenotypic signatures, which can hence be exploited to surmount the diagnostic and therapeutic challenges. Present review discusses the involvement of miRNAs in oncogenesis with the analysis of patented research available on miRNAs. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gupta, Tushar; Robles, Maria Teresa Sáenz; Schowalter, Rachel M.

Polyomaviruses induce cell proliferation and transformation through different oncoproteins encoded within the early region (ER): large T antigen (LT), small T antigen (sT) and, in some cases, additional components. Each virus utilizes different mechanisms to achieve transformation. For instance, the LTs of Simian virus 40 (SV40), BK and/or JC virus can induce transformation; but Merkel Cell Polyomavirus (MCPyV) requires expression of sT. Lymphotropic Papovavirus (LPV) is closely related to Human Polyomavirus 9 (HuPyV9) and, under similar conditions, mice expressing LPV.ER exhibit higher rates of tumor formation than mice expressing SV40.ER. We have investigated the contributions of individual LPV.ER components tomore » cell transformation. In contrast to SV40, LPV.ER transforms mouse embryonic fibroblasts (MEFs), but expression of LPV LT is insufficient to transform MEFs. Furthermore, LPV sT induces immortalization and transformation of MEFs. Thus, in the case of LPV, sT is the main mediator of oncogenesis. - Highlights: • Characterization of early region products from the Lymphotropic Polyomavirus (LPV). • On its own, sT immortalizes and transforms mouse primary cells, and is able to block p53 activation. • Combined LT and sT expression induces a greater rate of proliferation than either LT or sT alone.« less

Questioning the utility of pooling samples in microarray experiments with cell lines.

PubMed

Lusa, L; Cappelletti, V; Gariboldi, M; Ferrario, C; De Cecco, L; Reid, J F; Toffanin, S; Gallus, G; McShane, L M; Daidone, M G; Pierotti, M A

2006-01-01

We describe a microarray experiment using the MCF-7 breast cancer cell line in two different experimental conditions for which the same number of independent pools as the number of individual samples was hybridized on Affymetrix GeneChips. Unexpectedly, when using individual samples, the number of probe sets found to be differentially expressed between treated and untreated cells was about three times greater than that found using pools. These findings indicate that pooling samples in microarray experiments where the biological variability is expected to be small might not be helpful and could even decrease one's ability to identify differentially expressed genes.

Prohormone convertase and autocrine growth factor mRNAs are coexpressed in small cell lung carcinoma.

PubMed

Rounseville, M P; Davis, T P

2000-08-01

A hallmark of small cell lung carcinoma (SCLC) is the expression of autocrine growth factors such as neurotensin and gastrin-releasing peptide, which bind to cellular receptors and stimulate cell division. The biological activity of autocrine growth factors requires the concurrent expression of prohormone convertases that cleave the growth factors to their active form, suggesting the expression of these genes is linked in SCLCs. RNase protection assays were used to detect the expression of autocrine growth factor and prohormone convertase mRNAs in a panel of lung cancer cell lines. These mRNAs are coexpressed in SCLC and lung carcinoid cell lines, but not in normal lung epithelium or in non-small cell lung cancers. These findings, together with earlier results from our laboratory, suggest the expression of prohormone convertases has an important role in the development and maintenance of the SCLC phenotype and that autocrine growth factor and prohormone convertase genes respond to a common transcriptional activator in SCLC.

RNAi-Mediated Knockdown of IKK1 in Transgenic Mice Using a Transgenic Construct Containing the Human H1 Promoter

PubMed Central

Moreno-Maldonado, Rodolfo; Murillas, Rodolfo; Page, Angustias; Suarez-Cabrera, Cristian; Alameda, Josefa P.; Bravo, Ana; Casanova, M. Llanos

2014-01-01

Inhibition of gene expression through siRNAs is a tool increasingly used for the study of gene function in model systems, including transgenic mice. To achieve perdurable effects, the stable expression of siRNAs by an integrated transgenic construct is necessary. For transgenic siRNA expression, promoters transcribed by either RNApol II or III (such as U6 or H1 promoters) can be used. Relatively large amounts of small RNAs synthesis are achieved when using RNApol III promoters, which can be advantageous in knockdown experiments. To study the feasibility of H1 promoter-driven RNAi-expressing constructs for protein knockdown in transgenic mice, we chose IKK1 as the target gene. Our results indicate that constructs containing the H1 promoter are sensitive to the presence of prokaryotic sequences and to transgene position effects, similar to RNApol II promoters-driven constructs. We observed variable expression levels of transgenic siRNA among different tissues and animals and a reduction of up to 80% in IKK1 expression. Furthermore, IKK1 knockdown led to hair follicle alterations. In summary, we show that constructs directed by the H1 promoter can be used for knockdown of genes of interest in different organs and for the generation of animal models complementary to knockout and overexpression models. PMID:24523631

Intratumor Heterogeneity of ALK-Rearrangements and Homogeneity of EGFR-Mutations in Mixed Lung Adenocarcinoma

PubMed Central

Marino, Federica Zito; Liguori, Giuseppina; Aquino, Gabriella; La Mantia, Elvira; Bosari, Silvano; Ferrero, Stefano; Rosso, Lorenzo; Gaudioso, Gabriella; De Rosa, Nicla; Scrima, Marianna; Martucci, Nicola; La Rocca, Antonello; Normanno, Nicola; Morabito, Alessandro; Rocco, Gaetano; Botti, Gerardo; Franco, Renato

2015-01-01

Background Non Small Cell Lung Cancer is a highly heterogeneous tumor. Histologic intratumor heterogeneity could be ‘major’, characterized by a single tumor showing two different histologic types, and ‘minor’, due to at least 2 different growth patterns in the same tumor. Therefore, a morphological heterogeneity could reflect an intratumor molecular heterogeneity. To date, few data are reported in literature about molecular features of the mixed adenocarcinoma. The aim of our study was to assess EGFR-mutations and ALK-rearrangements in different intratumor subtypes and/or growth patterns in a series of mixed adenocarcinomas and adenosquamous carcinomas. Methods 590 Non Small Cell Lung Carcinomas tumor samples were revised in order to select mixed adenocarcinomas with available tumor components. Finally, only 105 mixed adenocarcinomas and 17 adenosquamous carcinomas were included in the study for further analyses. Two TMAs were built selecting the different intratumor histotypes. ALK-rearrangements were detected through FISH and IHC, and EGFR-mutations were detected through IHC and confirmed by RT-PCR. Results 10/122 cases were ALK-rearranged and 7 from those 10 showing an intratumor heterogeneity of the rearrangements. 12/122 cases were EGFR-mutated, uniformly expressing the EGFR-mutated protein in all histologic components. Conclusion Our data suggests that EGFR-mutations is generally homogeneously expressed. On the contrary, ALK-rearrangement showed an intratumor heterogeneity in both mixed adenocarcinomas and adenosquamous carcinomas. The intratumor heterogeneity of ALK-rearrangements could lead to a possible impact on the therapeutic responses and the disease outcomes. PMID:26422230

Vanilloid Receptor-1 (TRPV1) Expression and Function in the Vasculature of the Rat

PubMed Central

Czikora, Ágnes; Pásztor, Enikő T.; Dienes, Beatrix; Bai, Péter; Csernoch, László; Rutkai, Ibolya; Csató, Viktória; Mányiné, Ivetta S.; Pórszász, Róbert; Édes, István; Papp, Zoltán; Boczán, Judit

2014-01-01

Transient receptor potential (TRP) cation channels are emerging in vascular biology. In particular, the expression of the capsaicin receptor (TRPV1) was reported in vascular smooth muscle cells. This study characterized the arteriolar TRPV1 function and expression in the rat. TRPV1 mRNA was expressed in various vascular beds. Six commercially available antibodies were tested for TRPV1 specificity. Two of them were specific (immunostaining was abolished by blocking peptides) for neuronal TRPV1 and one recognized vascular TRPV1. TRPV1 was expressed in blood vessels in the skeletal muscle, mesenteric and skin tissues, as well as in the aorta and carotid arteries. TRPV1 expression was found to be regulated at the level of individual blood vessels, where some vessels expressed, while others did not express TRPV1 in the same tissue sections. Capsaicin (a TRPV1 agonist) evoked constrictions in skeletal muscle arteries and in the carotid artery, but had no effect on the femoral and mesenteric arteries or the aorta. In blood vessels, TRPV1 expression was detected in most of the large arteries, but there were striking differences at level of the small arteries. TRPV1 activity was suppressed in some isolated arteries. This tightly regulated expression and function suggests a physiological role for vascular TRPV1. PMID:24217926

psRNATarget: a plant small RNA target analysis server

PubMed Central

Dai, Xinbin; Zhao, Patrick Xuechun

2011-01-01

Plant endogenous non-coding short small RNAs (20–24 nt), including microRNAs (miRNAs) and a subset of small interfering RNAs (ta-siRNAs), play important role in gene expression regulatory networks (GRNs). For example, many transcription factors and development-related genes have been reported as targets of these regulatory small RNAs. Although a number of miRNA target prediction algorithms and programs have been developed, most of them were designed for animal miRNAs which are significantly different from plant miRNAs in the target recognition process. These differences demand the development of separate plant miRNA (and ta-siRNA) target analysis tool(s). We present psRNATarget, a plant small RNA target analysis server, which features two important analysis functions: (i) reverse complementary matching between small RNA and target transcript using a proven scoring schema, and (ii) target-site accessibility evaluation by calculating unpaired energy (UPE) required to ‘open’ secondary structure around small RNA’s target site on mRNA. The psRNATarget incorporates recent discoveries in plant miRNA target recognition, e.g. it distinguishes translational and post-transcriptional inhibition, and it reports the number of small RNA/target site pairs that may affect small RNA binding activity to target transcript. The psRNATarget server is designed for high-throughput analysis of next-generation data with an efficient distributed computing back-end pipeline that runs on a Linux cluster. The server front-end integrates three simplified user-friendly interfaces to accept user-submitted or preloaded small RNAs and transcript sequences; and outputs a comprehensive list of small RNA/target pairs along with the online tools for batch downloading, key word searching and results sorting. The psRNATarget server is freely available at http://plantgrn.noble.org/psRNATarget/. PMID:21622958

[Expression and clinical significance of CD147 in parathyroid carcinoma].

PubMed

Du, X M; Wang, L L; Chang, H; Meng, W; Zhang, J Y; Shen, B

2016-06-08

To study the expression and clinical significance of CD147 in the patients of parathyroid carcinoma. Fourteen cases of parathyroid carcinoma encountered during the period from 2012 to 2015 were enrolled. Thirty three cases of parathyroid adenoma encountered during the same period were enrolled. The expression of CD147 in parathyroid carcinoma and parathyroid adenoma was studied by means of immunohistochemistry (EnVision method). CD147 positive color was brown and yellow, and positive position was located mainly in the cytomembrane, and a small amount of cytoplasm was appeared. Among 14 cases of parathyroid carcinoma, 11 cases of CD147 positive score was 3+ , 3 cases of CD147 positive score was 2+ ; Among 33 cases of parathyroid adenoma , 8 cases of CD147 positive score was 2+ , 15 cases of it was 1+ , 10 cases of it was negative. CD147 was highly expressed in parathyroid carcinoma tissues, and the expression of CD147 was significantly different from the expression of parathyroid adenoma(P<0.05). CD147 immunohistochemical staining can help to diagnose parathyroid carcinoma.

Identification of miRNAs during mouse postnatal ovarian development and superovulation.

PubMed

Khan, Hamid Ali; Zhao, Yi; Wang, Li; Li, Qian; Du, Yu-Ai; Dan, Yi; Huo, Li-Jun

2015-07-08

MicroRNAs are small noncoding RNAs that play critical roles in regulation of gene expression in wide array of tissues including the ovary through sequence complementarity at post-transcriptional level. Tight regulation of multitude of genes involved in ovarian development and folliculogenesis could be regulated at transcription level by these miRNAs. Therefore, tissue specific miRNAs identification is considered a key step towards understanding the role of miRNAs in biological processes. To investigate the role of microRNAs during ovarian development and folliculogenesis we sequenced eight different libraries using Illumina deep sequencing technology. Different developmental stages were selected to explore miRNAs expression pattern at different stages of gonadal maturation with/without treatment of PMSG/hCG for superovulation. From massive sequencing reads, clean reads of 16-26 bp were selected for further analysis of differential expression analysis and novel microRNA annotation. Expression analysis of all miRNAs at different developmental stages showed that some miRNAs were present ubiquitously while others were differentially expressed at different stages. Among differentially expressed miRNAs we reported 61 miRNAs with a fold change of more than 2 at different developmental stages among all libraries. Among the up-regulated miRNAs, mmu-mir-1298 had the highest fold change with 4.025 while mmu-mir-150 was down-regulated more than 3 fold. Furthermore, we found 2659 target genes for 20 differentially expressed microRNAs using seven different target predictions programs (DIANA-mT, miRanda, miRDB, miRWalk, RNAhybrid, PICTAR5, TargetScan). Analysis of the predicted targets showed certain ovary specific genes targeted by single or multiple microRNAs. Furthermore, pathway annotation and Gene ontology showed involvement of these microRNAs in basic cellular process. These results suggest the presence of different miRNAs at different stages of ovarian development and superovulation. Potential role of these microRNAs was elucidated using bioinformatics tools in regulation of different pathways, biological functions and cellular components underlying ovarian development and superovulation. These results provide a framework for extended analysis of miRNAs and their roles during ovarian development and superovulation. Furthermore, this study provides a base for characterization of individual miRNAs to discover their role in ovarian development and female fertility.

Distinct RNAi Pathways in the Regulation of Physiology and Development in the Fungus Mucor circinelloides.

PubMed

Ruiz-Vázquez, Rosa M; Nicolás, Francisco E; Torres-Martínez, Santiago; Garre, Victoriano

2015-01-01

The basal fungus Mucor circinelloides has become, in recent years, a valuable model to study RNA-mediated gene silencing or RNA interference (RNAi). Serendipitously discovered in the late 1900s, the gene silencing in M. circinelloides is a landscape of consensus and dissents. Although similar to other classical fungal models in the basic design of the essential machinery that is responsible for silencing of gene expression, the existence of small RNA molecules of different sizes generated during this process and the presence of a mechanism that amplifies the silencing signal, give it a unique identity. In addition, M. circinelloides combines the components of RNAi machinery to carry out functions that not only limit themselves to the defense against foreign genetic material, but it uses some of these elements to regulate the expression of its own genes. Thus, different combinations of RNAi elements produce distinct classes of endogenous small RNAs (esRNAs) that regulate different physiological and developmental processes in response to environmental signals. The recent discovery of a new RNAi pathway involved in the specific degradation of endogenous mRNAs, using a novel RNase protein, adds one more element to the exciting puzzle of the gene silencing in M. circinelloides, in addition to providing hints about the evolutionary origin of the RNAi mechanism. Copyright © 2015 Elsevier Inc. All rights reserved.

BATS: a Bayesian user-friendly software for analyzing time series microarray experiments.

PubMed

Angelini, Claudia; Cutillo, Luisa; De Canditiis, Daniela; Mutarelli, Margherita; Pensky, Marianna

2008-10-06

Gene expression levels in a given cell can be influenced by different factors, namely pharmacological or medical treatments. The response to a given stimulus is usually different for different genes and may depend on time. One of the goals of modern molecular biology is the high-throughput identification of genes associated with a particular treatment or a biological process of interest. From methodological and computational point of view, analyzing high-dimensional time course microarray data requires very specific set of tools which are usually not included in standard software packages. Recently, the authors of this paper developed a fully Bayesian approach which allows one to identify differentially expressed genes in a 'one-sample' time-course microarray experiment, to rank them and to estimate their expression profiles. The method is based on explicit expressions for calculations and, hence, very computationally efficient. The software package BATS (Bayesian Analysis of Time Series) presented here implements the methodology described above. It allows an user to automatically identify and rank differentially expressed genes and to estimate their expression profiles when at least 5-6 time points are available. The package has a user-friendly interface. BATS successfully manages various technical difficulties which arise in time-course microarray experiments, such as a small number of observations, non-uniform sampling intervals and replicated or missing data. BATS is a free user-friendly software for the analysis of both simulated and real microarray time course experiments. The software, the user manual and a brief illustrative example are freely available online at the BATS website: http://www.na.iac.cnr.it/bats.

Differential expression of miRNAs in the nervous system of a rat model of bilateral sciatic nerve chronic constriction injury.

PubMed

Li, Haixia; Shen, Le; Ma, Chao; Huang, Yuguang

2013-07-01

Chronic neuropathic pain is associated with global changes in gene expression in different areas of the nociceptive pathway. MicroRNAs (miRNAs) are small (~22 nt long) non-coding RNAs, which are able to regulate hundreds of different genes post-transcriptionally. The aim of this study was to determine the miRNA expression patterns in the different regions of the pain transmission pathway using a rat model of human neuropathic pain induced by bilateral sciatic nerve chronic constriction injury (bCCI). Using microarray analysis and quantitative reverse transcriptase-PCR, we observed a significant upregulation in miR-341 expression in the dorsal root ganglion (DRG), but not in the spinal dorsal horn (SDH), hippocampus or anterior cingulate cortex (ACC), in the rats with neuropathic pain compared to rats in the naïve and sham-operated groups. By contrast, the expression of miR-203, miR-181a-1* and miR-541* was significantly reduced in the SDH of rats with neuropathic pain. Our data indicate that miR-341 is upregulated in the DRG, whereas miR-203, miR-181a-1* and miR-541* are downregulated in the SDH under neuropathic pain conditions. Thus, the differential expression of miRNAs in the nervous system may play a role in the development of chronic pain. These observations may aid in the development of novel treatment methods for neuropathic pain, which may involve miRNA gene therapy in local regions.

Multiscale mechanisms of nutritionally induced property variation in spider silks.

PubMed

Blamires, Sean J; Nobbs, Madeleine; Martens, Penny J; Tso, I-Min; Chuang, Wei-Tsung; Chang, Chung-Kai; Sheu, Hwo-Shuenn

2018-01-01

Variability in spider major ampullate (MA) silk properties at different scales has proven difficult to determine and remains an obstacle to the development of synthetic fibers mimicking MA silk performance. A multitude of techniques may be used to measure multiscale aspects of silk properties. Here we fed five species of Araneoid spider solutions that either contained protein or were protein deprived and performed silk tensile tests, small and wide-angle X-ray scattering (SAXS/WAXS), amino acid composition analyses, and silk gene expression analyses, to resolve persistent questions about how nutrient deprivation induces variations in MA silk mechanical properties across scales. Our analyses found that the properties of each spider's silk varied differently in response to variations in their protein intake. We found changes in the crystalline and non-crystalline nanostructures to play specific roles in inducing the property variations we found. Across treatment MaSp expression patterns differed in each of the five species. We found that in most species MaSp expression and amino acid composition variations did not conform with our predictions based on a traditional MaSp expression model. In general, changes to the silk's alanine and proline compositions influenced the alignment of the proteins within the silk's amorphous region, which influenced silk extensibility and toughness. Variations in structural alignment in the crystalline and non-crystalline regions influenced ultimate strength independent of genetic expression. Our study provides the deepest insights thus far into the mechanisms of how MA silk properties vary from gene expression to nanostructure formations to fiber mechanics. Such knowledge is imperative for promoting the production of synthetic silk fibers.

Human Endometrial DNA Methylome Is Cycle-Dependent and Is Associated With Gene Expression Regulation

PubMed Central

Houshdaran, Sahar; Zelenko, Zara; Irwin, Juan C.

2014-01-01

Human endometrium undergoes major gene expression changes, resulting in altered cellular functions in response to cyclic variations in circulating estradiol and progesterone, largely mediated by transcription factors and nuclear receptors. In addition to classic modulators, epigenetic mechanisms regulate gene expression during development in response to environmental factors and in some diseases and have roles in steroid hormone action. Herein, we tested the hypothesis that DNA methylation plays a role in gene expression regulation in human endometrium in different hormonal milieux. High throughput, genome-wide DNA methylation profiling of endometrial samples in proliferative, early secretory, and midsecretory phases revealed dynamic DNA methylation patterns with segregation of proliferative from secretory phase samples by unsupervised cluster analysis of differentially methylated genes. Changes involved different frequencies of gain and loss of methylation within or outside CpG islands. Comparison of changes in transcriptomes and corresponding DNA methylomes from the same samples revealed association of DNA methylation and gene expression in a number of loci, some important in endometrial biology. Human endometrial stromal fibroblasts treated in vitro with estradiol and progesterone exhibited DNA methylation changes in several genes observed in proliferative and secretory phase tissues, respectively. Taken together, the data support the observation that epigenetic mechanisms are involved in gene expression regulation in human endometrium in different hormonal milieux, adding endometrium to a small number of normal adult tissues exhibiting dynamic DNA methylation. The data also raise the possibility that the interplay between steroid hormone and methylome dynamics regulates normal endometrial functions and, if abnormal, may result in endometrial dysfunction and associated disorders. PMID:24877562

Bacterial expression of self-assembling peptide hydrogelators

NASA Astrophysics Data System (ADS)

Sonmez, Cem

For tissue regeneration and drug delivery applications, various architectures are explored to serve as biomaterial tools. Via de novo design, functional peptide hydrogel materials have been developed as scaffolds for biomedical applications. The objective of this study is to investigate bacterial expression as an alternative method to chemical synthesis for the recombinant production of self-assembling peptides that can form rigid hydrogels under physiological conditions. The Schneider and Pochan Labs have designed and characterized a 20 amino acid beta-hairpin forming amphiphilic peptide containing a D-residue in its turn region (MAX1). As a result, this peptide must be prepared chemically. Peptide engineering, using the sequence of MAX1 as a template, afforded a small family of peptides for expression (EX peptides) that have different turn sequences consisting of natural amino acids and amenable to bacterial expression. Each sequence was initially chemically synthesized to quickly assess the material properties of its corresponding gel. One model peptide EX1, was chosen to start the bacterial expression studies. DNA constructs facilitating the expression of EX1 were designed in such that the peptide could be expressed with different fusion partners and subsequently cleaved by enzymatic or chemical means to afford the free peptide. Optimization studies were performed to increase the yield of pure peptide that ultimately allowed 50 mg of pure peptide to be harvested from one liter of culture, providing an alternate means to produce this hydrogel-forming peptide. Recombinant production of other self-assembling hairpins with different turn sequences was also successful using this optimized protocol. The studies demonstrate that new beta-hairpin self-assembling peptides that are amenable to bacterial production and form rigid hydrogels at physiological conditions can be designed and produced by fermentation in good yield at significantly reduced cost when compared to chemical synthesis.

RNA sequencing of transformed lymphoblastoid cells from siblings discordant for autism spectrum disorders reveals transcriptomic and functional alterations: Evidence for sex-specific effects.

PubMed

Tylee, Daniel S; Espinoza, Alfred J; Hess, Jonathan L; Tahir, Muhammad A; McCoy, Sarah Y; Rim, Joshua K; Dhimal, Totadri; Cohen, Ori S; Glatt, Stephen J

2017-03-01

Genome-wide expression studies of samples derived from individuals with autism spectrum disorder (ASD) and their unaffected siblings have been widely used to shed light on transcriptomic differences associated with this condition. Females have historically been under-represented in ASD genomic studies. Emerging evidence from studies of structural genetic variants and peripheral biomarkers suggest that sex-differences may exist in the biological correlates of ASD. Relatively few studies have explicitly examined whether sex-differences exist in the transcriptomic signature of ASD. The present study quantified genome-wide expression values by performing RNA sequencing on transformed lymphoblastoid cell lines and identified transcripts differentially expressed between same-sex, proximal-aged sibling pairs. We found that performing separate analyses for each sex improved our ability to detect ASD-related transcriptomic differences; we observed a larger number of dysregulated genes within our smaller set of female samples (n = 12 sibling pairs), as compared with the set of male samples (n = 24 sibling pairs), with small, but statistically significant overlap between the sexes. Permutation-based gene-set analyses and weighted gene co-expression network analyses also supported the idea that the transcriptomic signature of ASD may differ between males and females. We discuss our findings in the context of the relevant literature, underscoring the need for future ASD studies to explicitly account for differences between the sexes. Autism Res 2017, 10: 439-455. © 2016 International Society for Autism Research, Wiley Periodicals, Inc. © 2016 International Society for Autism Research, Wiley Periodicals, Inc.

Radiation-induced interleukin-6 expression through MAPK/p38/NF-kappaB signaling pathway and the resultant antiapoptotic effect on endothelial cells through Mcl-1 expression with sIL6-Ralpha.

PubMed

Chou, Chia-Hung; Chen, Shee-Uan; Cheng, Jason Chia-Hsien

2009-12-01

To investigate the mechanism of interleukin-6 (IL-6) activity induced by ionizing radiation. Human umbilical vascular endothelial cells (HUVECs) were irradiated with different doses to induce IL-6. The IL-6 promoter assay and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to examine transcriptional regulation. Specific chemical inhibitors, decoy double-stranded oligodeoxynucleotides, and Western blotting were conducted to investigate the signal transduction pathway. Recombinant soluble human IL-6 receptor alpha-chain (sIL6-Ralpha) and specific small interfering RNA were used to evaluate the biologic function of radiation-induced IL-6. Four grays of radiation induced the highest level of IL-6 protein. The promoter assay and RT-PCR data revealed that the induction of IL-6 was mediated through transcriptional regulation. The p38 inhibitor SB203580, by blocking nuclear factor-kappaB (NF-kappaB) activation, prevented both the transcriptional and translational regulation of radiation-induced IL-6 expression. The addition of sIL6-Ralpha rescued HUVECs from radiation-induced death in an IL-6 concentratio-dependent manner. The antiapoptotic effect of combined sIL6-Ralpha and radiation-induced IL-6 was inhibited by mcl-1-specific small interfering RNA. Radiation transcriptionally induces IL-6 expression in endothelial cells through mitogen-activated protein kinase/p38-mediated NF-kappaB/IkappaB (inhibitor of NF-kappaB) complex activation. In the presence of sIL6-Ralpha, radiation-induced IL-6 expression acts through Mcl-1 expression to rescue endothelial cells from radiation-induced death.

Concomitant expression of far upstream element (FUSE) binding protein (FBP) interacting repressor (FIR) and its splice variants induce migration and invasion of non-small cell lung cancer (NSCLC) cells.

PubMed

Müller, Benedikt; Bovet, Michael; Yin, Yi; Stichel, Damian; Malz, Mona; González-Vallinas, Margarita; Middleton, Alistair; Ehemann, Volker; Schmitt, Jennifer; Muley, Thomas; Meister, Michael; Herpel, Esther; Singer, Stephan; Warth, Arne; Schirmacher, Peter; Drasdo, Dirk; Matthäus, Franziska; Breuhahn, Kai

2015-11-01

Transcription factors integrate a variety of oncogenic input information, facilitate tumour growth and cell dissemination, and therefore represent promising therapeutic target structures. Because over-expression of DNA-interacting far upstream element binding protein (FBP) supports non-small cell lung cancer (NSCLC) migration, we asked whether its repressor, FBP-interacting repressor (FIR) is functionally inactivated and how FIR might affect NSCLC cell biology. Different FIR splice variants were highly expressed in the majority of NSCLCs, with the highest levels in tumours carrying genomic gains of chromosome 8q24.3, which contained the FIR gene locus. Nuclear FIR expression was significantly enriched at the invasion front of primary NSCLCs, but this did not correlate with tumour cell proliferation. FIR accumulation was associated with worse patient survival and tumour recurrence; in addition, FIR over-expression significantly correlated with lymph node metastasis in squamous cell carcinomas (SCCs). In vitro, we applied newly developed methods and modelling approaches for the quantitative and time-resolved description of the pro-migratory and pro-invasive capacities of SCC cells. siRNA-mediated silencing of all FIR variants significantly reduced the speed and directional movement of tumour cells in all phases of migration. Furthermore, sprouting efficiency and single cell invasiveness were diminished following FIR inhibition. Interestingly, the silencing of FIR isoforms lacking exon 2 (FIR(Δexon2)) alone was sufficient to reduce lateral migration and invasion. In summary, by using scale-spanning data derived from primary human tissues, quantitative cellular analyses and mathematical modelling, we have demonstrated that concomitant over-expression of FIR and its splice variants drives NSCLC migration and dissemination. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Comparative analysis of miRNA expression during the development of insects of different metamorphosis modes and germ-band types.

PubMed

Ylla, Guillem; Piulachs, Maria-Dolors; Belles, Xavier

2017-10-11

Do miRNAs contribute to specify the germ-band type and the body structure in the insect embryo? Our goal was to address that issue by studying the changes in miRNA expression along the ontogeny of the German cockroach Blattella germanica, which is a short germ-band and hemimetabolan species. We sequenced small RNA libraries representing 11 developmental stages of B. germanica ontogeny (with especial emphasis on embryogenesis) and the changes in miRNA expression were examined. Data were compared with equivalent data for two long germ-band holometabolan species Drosophila melanogaster and Drosophila virilis, and the short germ-band holometabolan species Tribolium castaneum. The identification of B. germanica embryo small RNA sequences unveiled miRNAs not detected in previous studies, such as those of the MIR-309 family and 54 novel miRNAs. Four main waves of miRNA expression were recognized (with most miRNA changes occurring during the embryonic stages): the first from day 0 to day 1 of embryogenesis, the second during mid-embryogenesis (days 0-6), the third (with an acute expression peak) on day 2 of embryonic development, and the fourth during post-embryonic development. The second wave defined the boundaries of maternal-to-zygotic transition, with maternal mRNAs being cleared, presumably by Mir-309 and associated scavenger miRNAs. miRNAs follow well-defined patterns of expression over hemimetabolan ontogeny, patterns that are more diverse during embryonic development than during the nymphal stages. The results suggest that miRNAs play important roles in the developmental transitions between the embryonic stages of development (starting with maternal loading), during which they might influence the germ-band type and metamorphosis mode.

MiRNAs with Apoptosis Regulating Potential Are Differentially Expressed in Chronic Exercise-Induced Physiologically Hypertrophied Hearts

PubMed Central

Ramprasath, Tharmarajan; Kalpana, Krishnan

2015-01-01

Physiological cardiac hypertrophy is an adaptive mechanism, induced during chronic exercise. As it is reversible and not associated with cardiomyocyte death, it is considered as a natural tactic to prevent cardiac dysfunction and failure. Though, different studies revealed the importance of microRNAs (miRNAs) in pathological hypertrophy, their role during physiological hypertrophy is largely unexplored. Hence, this study is aimed at revealing the global expression profile of miRNAs during physiological cardiac hypertrophy. Chronic swimming protocol continuously for eight weeks resulted in induction of physiological hypertrophy in rats and histopathology revealed the absence of tissue damage, apoptosis or fibrosis. Subsequently, the total RNA was isolated and small RNA sequencing was executed. Analysis of small RNA reads revealed the differential expression of a large set of miRNAs during physiological hypertrophy. The expression profile of the significantly differentially expressed miRNAs was validated by qPCR. In silico prediction of target genes by miRanda, miRdB and TargetScan and subsequent qPCR analysis unraveled that miRNAs including miR-99b, miR-100, miR-19b, miR-10, miR-208a, miR-133, miR-191a, miR-22, miR-30e and miR-181a are targeting the genes that primarily regulate cell proliferation and cell death. Gene ontology and pathway mapping showed that the differentially expressed miRNAs and their target genes were mapped to apoptosis and cell death pathways principally via PI3K/Akt/mTOR and MAPK signaling. In summary, our data indicates that regulation of these miRNAs with apoptosis regulating potential can be one of the major key factors in determining pathological or physiological hypertrophy by controlling fibrosis, apoptosis and cell death mechanisms. PMID:25793527

Paternal poly (ADP-ribose) metabolism modulates retention of inheritable sperm histones and early embryonic gene expression.

PubMed

Ihara, Motomasa; Meyer-Ficca, Mirella L; Leu, N Adrian; Rao, Shilpa; Li, Fan; Gregory, Brian D; Zalenskaya, Irina A; Schultz, Richard M; Meyer, Ralph G

2014-05-01

To achieve the extreme nuclear condensation necessary for sperm function, most histones are replaced with protamines during spermiogenesis in mammals. Mature sperm retain only a small fraction of nucleosomes, which are, in part, enriched on gene regulatory sequences, and recent findings suggest that these retained histones provide epigenetic information that regulates expression of a subset of genes involved in embryo development after fertilization. We addressed this tantalizing hypothesis by analyzing two mouse models exhibiting abnormal histone positioning in mature sperm due to impaired poly(ADP-ribose) (PAR) metabolism during spermiogenesis and identified altered sperm histone retention in specific gene loci genome-wide using MNase digestion-based enrichment of mononucleosomal DNA. We then set out to determine the extent to which expression of these genes was altered in embryos generated with these sperm. For control sperm, most genes showed some degree of histone association, unexpectedly suggesting that histone retention in sperm genes is not an all-or-none phenomenon and that a small number of histones may remain associated with genes throughout the genome. The amount of retained histones, however, was altered in many loci when PAR metabolism was impaired. To ascertain whether sperm histone association and embryonic gene expression are linked, the transcriptome of individual 2-cell embryos derived from such sperm was determined using microarrays and RNA sequencing. Strikingly, a moderate but statistically significant portion of the genes that were differentially expressed in these embryos also showed different histone retention in the corresponding gene loci in sperm of their fathers. These findings provide new evidence for the existence of a linkage between sperm histone retention and gene expression in the embryo.

Paternal Poly (ADP-ribose) Metabolism Modulates Retention of Inheritable Sperm Histones and Early Embryonic Gene Expression

PubMed Central

Leu, N. Adrian; Rao, Shilpa; Li, Fan; Gregory, Brian D.; Zalenskaya, Irina A.; Schultz, Richard M.; Meyer, Ralph G.

2014-01-01

To achieve the extreme nuclear condensation necessary for sperm function, most histones are replaced with protamines during spermiogenesis in mammals. Mature sperm retain only a small fraction of nucleosomes, which are, in part, enriched on gene regulatory sequences, and recent findings suggest that these retained histones provide epigenetic information that regulates expression of a subset of genes involved in embryo development after fertilization. We addressed this tantalizing hypothesis by analyzing two mouse models exhibiting abnormal histone positioning in mature sperm due to impaired poly(ADP-ribose) (PAR) metabolism during spermiogenesis and identified altered sperm histone retention in specific gene loci genome-wide using MNase digestion-based enrichment of mononucleosomal DNA. We then set out to determine the extent to which expression of these genes was altered in embryos generated with these sperm. For control sperm, most genes showed some degree of histone association, unexpectedly suggesting that histone retention in sperm genes is not an all-or-none phenomenon and that a small number of histones may remain associated with genes throughout the genome. The amount of retained histones, however, was altered in many loci when PAR metabolism was impaired. To ascertain whether sperm histone association and embryonic gene expression are linked, the transcriptome of individual 2-cell embryos derived from such sperm was determined using microarrays and RNA sequencing. Strikingly, a moderate but statistically significant portion of the genes that were differentially expressed in these embryos also showed different histone retention in the corresponding gene loci in sperm of their fathers. These findings provide new evidence for the existence of a linkage between sperm histone retention and gene expression in the embryo. PMID:24810616

Expression profile of small RNAs in Acacia mangium secondary xylem tissue with contrasting lignin content - potential regulatory sequences in monolignol biosynthetic pathway

PubMed Central

2011-01-01

Background Lignin, after cellulose, is the second most abundant biopolymer accounting for approximately 15-35% of the dry weight of wood. As an important component during wood formation, lignin is indispensable for plant structure and defense. However, it is an undesirable component in the pulp and paper industry. Removal of lignin from cellulose is costly and environmentally hazardous process. Tremendous efforts have been devoted to understand the role of enzymes and genes in controlling the amount and composition of lignin to be deposited in the cell wall. However, studies on the impact of downregulation and overexpression of monolignol biosynthesis genes in model species on lignin content, plant fitness and viability have been inconsistent. Recently, non-coding RNAs have been discovered to play an important role in regulating the entire monolignol biosynthesis pathway. As small RNAs have critical functions in various biological process during wood formation, small RNA profiling is an important tool for the identification of complete set of differentially expressed small RNAs between low lignin and high lignin secondary xylem. Results In line with this, we have generated two small RNAs libraries from samples with contrasting lignin content using Illumina GAII sequencer. About 10 million sequence reads were obtained in secondary xylem of Am48 with high lignin content (41%) and a corresponding 14 million sequence reads were obtained in secondary xylem of Am54 with low lignin content (21%). Our results suggested that A. mangium small RNAs are composed of a set of 12 highly conserved miRNAs families found in plant miRNAs database, 82 novel miRNAs and a large proportion of non-conserved small RNAs with low expression levels. The predicted target genes of those differentially expressed conserved and non-conserved miRNAs include transcription factors associated with regulation of the lignin biosynthetic pathway genes. Some of these small RNAs play an important role in epigenetic silencing. Differential expression of the small RNAs between secondary xylem tissues with contrasting lignin content suggests that a cascade of miRNAs play an interconnected role in regulating the lignin biosynthetic pathway in Acacia species. Conclusions Our study critically demonstrated the roles of small RNAs during secondary wall formation. Comparison of the expression pattern of small RNAs between secondary xylem tissues with contrasting lignin content strongly indicated that small RNAs play a key regulatory role during lignin biosynthesis. Our analyses suggest an evolutionary mechanism for miRNA targets on the basis of the length of their 5’ and 3’ UTRs and their cellular roles. The results obtained can be used to better understand the roles of small RNAs during lignin biosynthesis and for the development of gene constructs for silencing of specific genes involved in monolignol biosynthesis with minimal effect on plant fitness and viability. For the first time, small RNAs were proven to play an important regulatory role during lignin biosynthesis in A. mangium. PMID:22369296

Expression profile of small RNAs in Acacia mangium secondary xylem tissue with contrasting lignin content - potential regulatory sequences in monolignol biosynthetic pathway.

PubMed

Ong, Seong Siang; Wickneswari, Ratnam

2011-11-30

Lignin, after cellulose, is the second most abundant biopolymer accounting for approximately 15-35% of the dry weight of wood. As an important component during wood formation, lignin is indispensable for plant structure and defense. However, it is an undesirable component in the pulp and paper industry. Removal of lignin from cellulose is costly and environmentally hazardous process. Tremendous efforts have been devoted to understand the role of enzymes and genes in controlling the amount and composition of lignin to be deposited in the cell wall. However, studies on the impact of downregulation and overexpression of monolignol biosynthesis genes in model species on lignin content, plant fitness and viability have been inconsistent. Recently, non-coding RNAs have been discovered to play an important role in regulating the entire monolignol biosynthesis pathway. As small RNAs have critical functions in various biological process during wood formation, small RNA profiling is an important tool for the identification of complete set of differentially expressed small RNAs between low lignin and high lignin secondary xylem. In line with this, we have generated two small RNAs libraries from samples with contrasting lignin content using Illumina GAII sequencer. About 10 million sequence reads were obtained in secondary xylem of Am48 with high lignin content (41%) and a corresponding 14 million sequence reads were obtained in secondary xylem of Am54 with low lignin content (21%). Our results suggested that A. mangium small RNAs are composed of a set of 12 highly conserved miRNAs families found in plant miRNAs database, 82 novel miRNAs and a large proportion of non-conserved small RNAs with low expression levels. The predicted target genes of those differentially expressed conserved and non-conserved miRNAs include transcription factors associated with regulation of the lignin biosynthetic pathway genes. Some of these small RNAs play an important role in epigenetic silencing. Differential expression of the small RNAs between secondary xylem tissues with contrasting lignin content suggests that a cascade of miRNAs play an interconnected role in regulating the lignin biosynthetic pathway in Acacia species. Our study critically demonstrated the roles of small RNAs during secondary wall formation. Comparison of the expression pattern of small RNAs between secondary xylem tissues with contrasting lignin content strongly indicated that small RNAs play a key regulatory role during lignin biosynthesis. Our analyses suggest an evolutionary mechanism for miRNA targets on the basis of the length of their 5' and 3' UTRs and their cellular roles. The results obtained can be used to better understand the roles of small RNAs during lignin biosynthesis and for the development of gene constructs for silencing of specific genes involved in monolignol biosynthesis with minimal effect on plant fitness and viability. For the first time, small RNAs were proven to play an important regulatory role during lignin biosynthesis in A. mangium.

[Effect of Different Stimulating Strength of Electroacupuncture on Gastrointestinal Motility and RhoA/ROCK Signaling in Gastric Antral Smooth Muscle in Diabetic Gastroparesis Rats].

PubMed

Wu, Xue-Fen; Chen, Xiao-Li; Zheng, Xue-Na; Guo, Xin; Xie, Zhi-Qiang; Liu, Li; Wei, Xin-Ran; Yue, Zeng-Hui

2018-03-25

To observe the effect of different strength of electroacupuncture (EA) stimulation on gastrointestinal motility and Ras homolog gene family member (RhoA)/Rho associated coiled-coil forming protein kinase (ROCK) signaling in diabetic gastroparesis (DGP) rats, so as to reveal the underlying mechanisms of EA for improving DGP. Sixty SD rats were randomly and equally divided into blank control, DGP model, weak EA, medium EA, and strong EA groups ( n ＝12 rats in each). The DGP model was established by intraperitoneal injection of streptozotocin (STZ, 55 mmol/kg, 2%) and high-sugar and high-fat fodder feeding for 8 weeks. EA (0.12, 0.24, 0.36 mA, 20 Hz/100 Hz) was applied to "Zusanli" (ST 36), "Sanyinjiao" (SP 6) and "Liangmen" (ST 21) for 20 min, once daily for 15 successive days. Blood glucose levels were measured weekly with blood glucose meter and blood glucose test paper. Fecal phenol red excretion method was used to display gastric emptying and small intestinal propulsion function. The expression of RhoA protein in the gastric antral smooth muscle tissue was detected by immunohistochemistry and Western blot (WB), separately, and that of ROCK, myosin phosphatase target subunit 1 (MYPT 1) and phosphorylated (p)-MYPT 1 proteins in gastric antrum detected by WB. Compared with the blank control group, the gastric emptying rate and small intestine propulsion rate of the model group were significantly decreased ( P <0.05), and the blood glucose level was remarkably increased ( P <0.05). Moreover, the expression levels of RhoA, ROCK, MYPT 1 and p-MYPT 1 proteins in the gastric antrum were significantly down-regulated relevant to the control group ( P <0.05). After administration of EA, the decreased gastric emptying rate and intestinal propulsion rate, and the down-regulated expression of RhoA, ROCK, MYPT 1 and p-MYPT 1 proteins were significantly increased in the strong, medium and weak EA stimulation groups ( P <0.05). Comparison among the 3 EA groups showed that the strong stimulation was significantly superior to weak stimulation in up-regulating the expression of RhoA, ROCK, MYPT 1 and p-MYPT 1 proteins, and obviously superior to the medium stimulation in up-regulating RhoA and MYPT 1 protein levels ( P <0.05), while the medium stimulation was significantly stronger than the weak stimulation in up-regulating the expression of ROCK, MYPT 1 and p-MYPT 1 proteins ( P <0.05). There were no significant differences among the 3 EA groups in up-regulating the gastric emptying rate and small intestinal propulsion rate, and between the strong stimulation and medium stimulation in the expression levels of ROCK and p-MYPT 1 proteins ( P >0.05). Electroacupuncture stimulation of ST 36-SP 6-ST 21 at 0.12, 0.24 and 0.36 mA can promote the gastrointestinal motility in DGP rats, which may be associated with its effects in enhancing RhoA/ROCK signaling in the gastric antral smooth muscle at different degrees.

Aberrant Gene Expression in Dogs with Portosystemic Shunts

PubMed Central

Grinwis, Guy C. M.; Kummeling, Anne; van Gils, Ingrid H. M.; Koerkamp, Marian J. A. Groot.; van Leenen, Dik; Holstege, Frank C. P.; Penning, Louis C.; Rothuizen, Jan; Leegwater, Peter A. J.; Spee, Bart

2013-01-01

Congenital portosystemic shunts are developmental anomalies of the splanchnic vascular system that cause portal blood to bypass the liver. Large-breed dogs are predisposed for intrahepatic portosystemic shunts (IHPSS) and small-breed dogs for extrahepatic portosystemic shunts (EHPSS). While the phenotype resulting from portal bypass of the liver of the two types of shunt is identical, the genotype and molecular pathways involved are probably different. The aim of this study was to gain insight into the pathways involved in the different types of portosystemic shunting. Microarray analysis of mRNA expression in liver tissue from dogs with EHPSS and IHPSS revealed that the expression of 26 genes was altered in either IHPSS or EHPSS samples compared with that in liver samples from control dogs. Quantitative real-time PCR of these genes in 14 IHPSS, 17 EHPSS, and 8 control liver samples revealed a significant differential expression of ACBP, CCBL1, GPC3, HAMP, PALLD, VCAM1, and WEE1. Immunohistochemistry and Western blotting confirmed an increased expression of VCAM1 in IHPSS but its absence in EHPSS, an increased WEE1 expression in IHPSS but not in EHPSS, and a decreased expression of CCBL1 in both shunt types. Regarding their physiologic functions, these findings may indicate a causative role for VCAM1 in IHPSS and WEE1 for IHPSS. CCBL1 could be an interesting candidate to study not yet elucidated aspects in the pathophysiology of hepatic encephalopathy. PMID:23451256

Analysis of Pax6 contiguous gene deletions in the mouse, Mus musculus, identifies regions distinct from Pax6 responsible for extreme small-eye and belly-spotting phenotypes.

PubMed

Favor, Jack; Bradley, Alan; Conte, Nathalie; Janik, Dirk; Pretsch, Walter; Reitmeir, Peter; Rosemann, Michael; Schmahl, Wolfgang; Wienberg, Johannes; Zaus, Irmgard

2009-08-01

In the mouse Pax6 function is critical in a dose-dependent manner for proper eye development. Pax6 contiguous gene deletions were shown to be homozygous lethal at an early embryonic stage. Heterozygotes express belly spotting and extreme microphthalmia. The eye phenotype is more severe than in heterozygous Pax6 intragenic null mutants, raising the possibility that deletions are functionally different from intragenic null mutations or that a region distinct from Pax6 included in the deletions affects eye phenotype. We recovered and identified the exact regions deleted in three new Pax6 deletions. All are homozygous lethal at an early embryonic stage. None express belly spotting. One expresses extreme microphthalmia and two express the milder eye phenotype similar to Pax6 intragenic null mutants. Analysis of Pax6 expression levels and the major isoforms excluded the hypothesis that the deletions expressing extreme microphthalmia are directly due to the action of Pax6 and functionally different from intragenic null mutations. A region distinct from Pax6 containing eight genes was identified for belly spotting. A second region containing one gene (Rcn1) was identified for the extreme microphthalmia phenotype. Rcn1 is a Ca(+2)-binding protein, resident in the endoplasmic reticulum, participates in the secretory pathway and expressed in the eye. Our results suggest that deletion of Rcn1 directly or indirectly contributes to the eye phenotype in Pax6 contiguous gene deletions.

MicroRNA Expression Profile in Penile Cancer Revealed by Next-Generation Small RNA Sequencing

PubMed Central

Zhang, Yuanwei; Xu, Bo; Zhou, Jun; Fan, Song; Hao, Zongyao; Shi, Haoqiang; Zhang, Xiansheng; Kong, Rui; Xu, Lingfan; Gao, Jingjing; Zou, Duohong; Liang, Chaozhao

2015-01-01

Penile cancer (PeCa) is a relatively rare tumor entity but possesses higher morbidity and mortality rates especially in developing countries. To date, the concrete pathogenic signaling pathways and core machineries involved in tumorigenesis and progression of PeCa remain to be elucidated. Several studies suggested miRNAs, which modulate gene expression at posttranscriptional level, were frequently mis-regulated and aberrantly expressed in human cancers. However, the miRNA profile in human PeCa has not been reported before. In this present study, the miRNA profile was obtained from 10 fresh penile cancerous tissues and matched adjacent non-cancerous tissues via next-generation sequencing. As a result, a total of 751 and 806 annotated miRNAs were identified in normal and cancerous penile tissues, respectively. Among which, 56 miRNAs with significantly different expression levels between paired tissues were identified. Subsequently, several annotated miRNAs were selected randomly and validated using quantitative real-time PCR. Compared with the previous publications regarding to the altered miRNAs expression in various cancers and especially genitourinary (prostate, bladder, kidney, testis) cancers, the most majority of deregulated miRNAs showed the similar expression pattern in penile cancer. Moreover, the bioinformatics analyses suggested that the putative target genes of differentially expressed miRNAs between cancerous and matched normal penile tissues were tightly associated with cell junction, proliferation, growth as well as genomic instability and so on, by modulating Wnt, MAPK, p53, PI3K-Akt, Notch and TGF-β signaling pathways, which were all well-established to participate in cancer initiation and progression. Our work presents a global view of the differentially expressed miRNAs and potentially regulatory networks of their target genes for clarifying the pathogenic transformation of normal penis to PeCa, which research resource also provides new insights into future investigations aimed to explore the in-depth mechanisms of miRNAs and other small RNAs including piRNAs in penile carcinogenesis regulation and effective target-specific theragnosis. PMID:26158897

Expression Analysis of Previously Verified Fecal and Plasma Dow-regulated MicroRNAs (miR-4478, 1295-3p, 142-3p and 26a-5p), in FFPE Tissue Samples of CRC Patients.

PubMed

Ghanbari, Reza; Rezasoltani, Sama; Hashemi, Javad; Mohamadkhani, Ashraf; Tahmasebifar, Arash; Arefian, Ehsan; Mobarra, Naser; Asadi, Jahanbakhsh; Nazemalhosseini Mojarad, Ehsan; Yazdani, Yaghoub; Knuutila, Sakari; Malekzadeh, Reza

2017-02-01

Colorectal cancer (CRC) is one of the most common causes of cancer-related mortality worldwide. Early diagnosis of this neoplasm is critical and may reduce patients' mortality. MicroRNAs are small non-coding RNA molecules whose expression pattern can be altered in various diseases such as CRC. In this study, we evaluated the expression levels of miR-142-3p, miR-26a-5p (their reduced expression in plasma samples of CRC patients was previously confirmed), miR-4478 and miR-1295-3p (their reduced expression in stool samples of CRC patients was previously confirmed) in tissue samples of CRC patients in comparison to healthy subjects. To achieve this purpose, total RNA including small RNA was extracted from 53 CRC and 35 normal subjects' Formalin-fixed, Paraffin-embedded (FFPE) tissue samples using the miRNeasy FFPE Mini Kit. The expression levels of these four selected miRNAs were measured using quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR). We found that the expression levels of miR-4478 and miR-1295b-3p (two previously down-regulated fecal miRNAs) were significantly decreased in FFPE samples of CRC patients compared to healthy controls. On the other hand, no significant differences were seen in expression levels of miR-142-3p and miR-26a-5p (two previously down-regulated circulating miRNAs) in FFPE samples between these two groups. Regarding current findings, it may be concluded that to diagnose CRC patients based on the miRNAs approach, stool samples are more likely preferable to plasma samples; nevertheless, additional studies with more samples are needed to confirm the results.

Reduced Smad4 expression and DNA topoisomerase inhibitor chemosensitivity in non-small cell lung cancer.

PubMed

Ziemke, Michael; Patil, Tejas; Nolan, Kyle; Tippimanchai, Darinee; Malkoski, Stephen P

2017-07-01

Smad4 is a tumor suppressor that transduces transforming growth factor beta signaling and regulates genomic stability. We previously found that Smad4 knockdown in vitro inhibited DNA repair and increased sensitivity to DNA topoisomerase inhibitors. In this study, we assessed the association between reduced Smad4 expression and DNA topoisomerase inhibitor sensitivity in human non-small cell lung cancer (NSCLC) patients and evaluated the relationship between genomic alterations of Smad4 and molecular alterations in DNA repair molecules. We retrospectively identified NSCLC patients who received etoposide or gemcitabine. Chemotherapeutic response was quantified by RECIST 1.1 criteria and Smad4 expression was assessed by immunohistochemistry. Relationships between Smad4 mutation and DNA repair molecule mutations were evaluated using publically available datasets. We identified 28 individuals who received 30 treatments with gemcitabine or etoposide containing regimens for NSCLC. Reduced Smad4 expression was seen in 13/28 patients and was not associated with significant differences in clinical or pathologic parameters. Patients with reduced Smad4 expression had a larger response to DNA topoisomerase inhibitor containing regimens then patients with high Smad4 expression (-25.7% vs. -6.8% in lesion size, p=0.03); this relationship was more pronounced with gemcitabine containing regimens. The overall treatment response was higher in patients with reduced Smad4 expression (8/14 vs 2/16 p=0.02). Analysis of data from The Cancer Genome Atlas revealed that Smad4 mutation or homozygous loss was mutually exclusive with genomic alterations in DNA repair molecules. Reduced Smad4 expression may predict responsiveness to regimens that contain DNA topoisomerase inhibitors. That Smad4 signaling alterations are mutually exclusive with alterations in DNA repair machinery is consistent with an important role of Smad4 in regulating DNA repair. Copyright © 2017 Elsevier B.V. All rights reserved.

Upregulation of Long Noncoding RNA Small Nucleolar RNA Host Gene 18 Promotes Radioresistance of Glioma by Repressing Semaphorin 5A

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Rong; Department of Radiation Oncology, Fujian Medical University Union Hospital, Fuzhou, Fujian; Yao, Qiwei

Purpose: Although increasing evidence has shown that long noncoding RNAs play an important regulatory role in carcinogenesis and tumor progression, little is known about the role of small nucleolar RNA host gene 18 (SNHG18) in cancer. The goal of this study was to investigate the expression of SNHG18 and its clinical significance in glioma. Methods and Materials: Differences in the lncRNA expression profile between M059K and M059J cells were assessed by lncRNA expression microarray analysis. The expression and localization of SNHG18 in glioma cells or tissues was evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and in situ hybridization (ISH),more » respectively. the clinical associations of SNHG18 in glioma was evaluated by qRT-PCR, ISH and immunohistochemistry. The role of SNHG18 in glioma radiosensitivity was evaluated by colony formation assays, immunofluorescence, Western blot and tumor growth inhibition study. Results: The present study investigated the clinical associations of SNHG18 and its role in glioma. Our results showed that the expression of SNHG18 was remarkably upregulated in clinical glioma tissues compared with normal brain tissues. SNHG18 expression was associated with the clinical tumor grade and correlated negatively with isocitrate dehydrogenase 1 mutation. In addition, knockdown of SNHG18 with short hairpin RNA suppressed the radioresistance of glioma cells, and transgenic expression of SNHG18 had the opposite effect. Furthermore, xenograft tumors grown from cells with SNHG18 deletion were more radiosensitive than tumors grown from control cells. Further studies revealed that SNHG18 promotes radioresistance by inhibiting semaphorin 5A and that inhibition of semaphorin 5A expression abrogated the radiosensitizing effect caused by SNHG18 deletion. Conclusions: Our findings provide new insights into the role of SNHG18 in glioma and suggest its potential as a target for glioma therapy.« less

Selection and validation of reference genes for miRNA expression studies during porcine pregnancy.

PubMed

Wessels, Jocelyn M; Edwards, Andrew K; Zettler, Candace; Tayade, Chandrakant

2011-01-01

MicroRNAs comprise a family of small non-coding RNAs that modulate several developmental and physiological processes including pregnancy. Their ubiquitous presence is confirmed in mammals, worms, flies and plants. Although rapid advances have been made in microRNA research, information on stable reference genes for validation of microRNA expression is still lacking. Real time PCR is a widely used tool to quantify gene transcripts. An appropriate reference gene must be chosen to minimize experimental error in this system. A small difference in miRNA levels between experimental samples can be biologically meaningful as these entities can affect multiple targets in a pathway. This study examined the suitability of six commercially available reference genes (RNU1A, RNU5A, RNU6B, SNORD25, SCARNA17, and SNORA73A) in maternal-fetal tissues from healthy and spontaneously arresting/dying conceptuses from sows were separately analyzed at gestation day 20. Comparisons were also made with non-pregnant endometrial tissues from sows. Spontaneous fetal loss is a prime concern to the commercial pork industry. Our laboratory has previously identified deficits in vasculature development at maternal-fetal interface as one of the major participating causes of fetal loss. Using this well-established model, we have extended our studies to identify suitable microRNA reference genes. A methodical approach to assessing suitability was adopted using standard curve and melting curve analysis, PCR product sequencing, real time PCR expression in a panel of gestational tissues, and geNorm and NormFinder analysis. Our quantitative real time PCR analysis confirmed expression of all 6 reference genes in maternal and fetal tissues. All genes were uniformly expressed in tissues from healthy and spontaneously arresting conceptus attachment sites. Comparisons between tissue types (maternal/fetal/non-pregnant) revealed significant differences for RNU5A, RNU6B, SCARNA17, and SNORA73A expression. Based on our methodical assessment of all 6 reference genes, results suggest that RNU1A is the most stable reference gene for porcine pregnancy studies.

Effect of postharvest temperature and ethylene on carotenoid accumulation in the Flavedo and juice sacs of Satsuma Mandarin ( Citrus unshiu Marc.) fruit.

PubMed

Matsumoto, Hikaru; Ikoma, Yoshinori; Kato, Masaya; Nakajima, Naoko; Hasegawa, Yoshinori

2009-06-10

The effect of postharvest temperature (5, 20, and 30 degrees C) and ethylene at different temperatures (20 and 5 degrees C) on carotenoid content and composition and on the expression of the carotenoid biosynthesis-related genes was investigated in the flavedo and juice sacs of Satsuma mandarin ( Citrus unshiu Marc.) fruit. Under an ethylene-free atmosphere, storage at 20 degrees C rapidly increased the carotenoid content in flavedo and maintained the content in juice sacs. In contrast, storage at 5 and 30 degrees C gradually decreased the content in juice sacs but slowly increased that in flavedo. Under an ethylene atmosphere, storage at 20 degrees C enhanced the carotenoid accumulation in flavedo more dramatically than found under an ethylene-free atmosphere with distinct changes in the carotenoid composition but did not noticeably change the content and composition in juice sacs. In contrast, storage at 5 degrees C under an ethylene atmosphere repressed carotenoid accumulation with changes in the carotenoid composition in flavedo but did not clearly change the carotenoid content in juice sacs. Under an ethylene-free atmosphere, differences in the gene expression profile among the temperatures were observed but were not well-correlated with those in the carotenoid content in flavedo and juice sacs. Under an ethylene atmosphere, in flavedo, the gene expression of phytoene synthase (PSY) and phytoene desaturase (PDS) was slightly higher at 20 degrees C but lower at 5 degrees C than under an ethylene-free atmosphere. At 20 degrees C, the gene expression of several carotenoid biosynthetic enzymes promoted by ethylene seemed to be responsible for the enhanced accumulation of carotenoid in flavedo. In contrast, at 5 degrees C, the repressed gene expression of PSY and PDS by ethylene seemed to be primarily responsible for the repressed accumulation of carotenoid in flavedo. In juice sacs, the small response of the gene expression to ethylene seemed to be responsible for small changes in carotenoid accumulation under an ethylene atmosphere.

Assessment of Dental Fluorosis in Mmp20+/− Mice

PubMed Central

Sharma, R.; Tye, C.E.; Arun, A.; MacDonald, D.; Chatterjee, A.; Abrazinski, T.; Everett, E.T.; Whitford, G.M.; Bartlett, J.D.

2011-01-01

The molecular mechanisms that underlie dental fluorosis are poorly understood. The retention of enamel proteins hallmarking fluorotic enamel may result from impaired hydrolysis and/or removal of enamel proteins. Previous studies have suggested that partial inhibition of Mmp20 expression is involved in the etiology of dental fluorosis. Here we ask if mice expressing only one functional Mmp20 allele are more susceptible to fluorosis. We demonstrate that Mmp20+/− mice express approximately half the amount of MMP20 as do wild-type mice. The Mmp20 heterozygous mice have normal-appearing enamel, with Vickers microhardness values similar to those of wild-type control enamel. Therefore, reduced MMP20 expression is not solely responsible for dental fluorosis. With 50-ppm-fluoride (F−) treatment ad libitum, the Mmp20+/− mice had F− tissue levels similar to those of Mmp20+/+ mice. No significant difference in enamel hardness was observed between the F−-treated heterozygous and wild-type mice. Interestingly, we did find a small but significant difference in quantitative fluorescence between these two groups, which may be attributable to slightly higher protein content in the Mmp20+/− mouse enamel. We conclude that MMP20 plays a nominal role in dental enamel fluorosis. PMID:21386097

Identification of small non-coding RNA classes expressed in swine whole blood during HP-PRRSV infection

USDA-ARS?s Scientific Manuscript database

It has been established that reduced susceptibility to porcine reproductive and respiratory syndrome virus (PRRSV) has a genetic component. This genetic component may take the form of small non-coding RNAs (sncRNA), which are molecules that function as regulators of gene expression. Various sncRNAs ...

Small RNAs, big impact: small RNA pathways in transposon control and their effect on the host stress response.

PubMed

Wheeler, Bayly S

2013-12-01

Transposons are mobile genetic elements that are a major constituent of most genomes. Organisms regulate transposable element expression, transposition, and insertion site preference, mitigating the genome instability caused by uncontrolled transposition. A recent burst of research has demonstrated the critical role of small non-coding RNAs in regulating transposition in fungi, plants, and animals. While mechanistically distinct, these pathways work through a conserved paradigm. The presence of a transposon is communicated by the presence of its RNA or by its integration into specific genomic loci. These signals are then translated into small non-coding RNAs that guide epigenetic modifications and gene silencing back to the transposon. In addition to being regulated by the host, transposable elements are themselves capable of influencing host gene expression. Transposon expression is responsive to environmental signals, and many transposons are activated by various cellular stresses. TEs can confer local gene regulation by acting as enhancers and can also confer global gene regulation through their non-coding RNAs. Thus, transposable elements can act as stress-responsive regulators that control host gene expression in cis and trans.

The C. elegans CSR-1 Argonaute pathway counteracts epigenetic silencing to promote germline gene expression

PubMed Central

Seth, Meetu; Shirayama, Masaki; Gu, Weifeng; Ishidate, Takao; Conte, Darryl; Mello, Craig C.

2014-01-01

SUMMARY Organisms can develop adaptive sequence-specific immunity by re-expressing pathogen-specific small RNAs that guide gene silencing. For example, the C. elegans PIWI-Argonaute/piRNA pathway recruits RNA-dependent RNA polymerase RdRP to foreign sequences to amplify a trans-generational small RNA-induced epigenetic silencing signal (termed RNAe). Here we provide evidence that in addition to an adaptive memory of silenced sequences, C. elegans can also develop an opposing adaptive memory of expressed/self mRNAs. We refer to this mechanism, which can prevent or reverse RNAe as RNA-induced epigenetic gene activation (RNAa). We show that CSR-1, which engages RdRP-amplified small RNAs complementary to germline-expressed mRNAs, is required for RNAa. We show that a transgene with RNAa activity also exhibits accumulation of cognate CSR-1 small RNAs. Our findings suggest that C. elegans adaptively acquires and maintains a trans-generational CSR-1 memory that recognizes and protects self mRNAs, allowing piRNAs to recognize foreign sequences innately, without need for prior exposure. PMID:24360782

quenched-smFISH: Counting small RNA in Pathogenic Bacteria

NASA Astrophysics Data System (ADS)

Shepherd, Douglas; Li, Nan; Micheva-Viteva, Sofiya; Munsky, Brian; Hong-Geller, Elizabeth; Werner, James

2014-03-01

Here, we present a modification to single-molecule fluorescence in situ hybridization, quenched smFISH (q-smFISH), that enables quantitative detection and analysis of small RNA (sRNA) expressed in bacteria. We show that short nucleic acid targets can be detected when the background of unbound singly dye-labeled DNA oligomers is reduced through hybridization with a set of complementary DNA oligomers labeled with a fluorescence quencher. Exploiting an automated, multi-color wide-field microscope and GPU-accelerated data analysis package, we analyzed the statistics of sRNA expression in thousands of individual Yersinia pseudotuberculosis and Yersinia pestis bacteria before and during a simulated infection. Before infection, we find only a small fraction of either bacteria express the small RNAs YSR35 or YSP8. The copy numbers of these RNA are increased during simulated infection, suggesting a role in pathogenesis. The ability to directly quantify expression level changes of sRNA in single cells as a function of external stimuli provides key information on the role of sRNA in bacterial regulatory networks.

Alterations in the small intestinal wall and motor function after repeated cisplatin in rat.

PubMed

Uranga, J A; García-Martínez, J M; García-Jiménez, C; Vera, G; Martín-Fontelles, M I; Abalo, R

2017-07-01

Gastrointestinal adverse effects occurring during cancer chemotherapy are well known and feared; those persisting once treatment has finished are relatively unknown. We characterized the alterations occurring in the rat small intestine, after repeated treatment with cisplatin. Male Wistar rats received saline or cisplatin (2 mg kg -1  week -1 , for 5 weeks, ip). Gastric motor function was studied non-invasively throughout treatment (W1-W5) and 1 week after treatment finalization (W6). During W6, upper gastrointestinal motility was also invasively studied and small intestinal samples were collected for histopathological and molecular studies. Structural alterations in the small intestinal wall, mucosa, submucosa, muscle layers, and lymphocytic nodules were histologically studied. Periodic acid-Schiff staining and immunohistochemistry for Ki-67, chromogranin A, and neuronal-specific enolase were used to detect secretory, proliferating, endocrine and neural cells, respectively. The expression of different markers in the tunica muscularis was analyzed by RT/qPCR. Repeated cisplatin induced motility alterations during and after treatment. After treatment (W6), the small intestinal wall showed histopathological alterations in most parameters measured, including a reduction in the thickness of circular and longitudinal muscle layers. Expression of c-KIT (for interstitial cells of Cajal), nNOS (for inhibitory motor neurons), pChAT, and cChAT (for excitatory motor neurons) increased significantly (although both ChATs to a lesser extent). Repeated cisplatin induces relatively long-lasting gut dysmotility in rat associated with important histopathological and molecular alterations in the small intestinal wall. In cancer survivors, the possible chemotherapy-induced histopathological, molecular, and functional intestinal sequelae should be evaluated. © 2017 John Wiley & Sons Ltd.

Endometrial gene expression profile of pregnant sows with extreme phenotypes for reproductive efficiency.

PubMed

Córdoba, S; Balcells, I; Castelló, A; Ovilo, C; Noguera, J L; Timoneda, O; Sánchez, A

2015-10-05

Prolificacy can directly impact porcine profitability, but large genetic variation and low heritability have been found regarding litter size among porcine breeds. To identify key differences in gene expression associated to swine reproductive efficiency, we performed a transcriptome analysis of sows' endometrium from an Iberian x Meishan F2 population at day 30-32 of gestation, classified according to their estimated breeding value (EBV) as high (H, EBV > 0) and low (L, EBV < 0) prolificacy phenotypes. For each sample, mRNA and small RNA libraries were RNA-sequenced, identifying 141 genes and 10 miRNAs differentially expressed between H and L groups. We selected four miRNAs based on their role in reproduction, and five genes displaying the highest differences and a positive mapping into known reproductive QTLs for RT-qPCR validation on the whole extreme population. Significant differences were validated for genes: PTGS2 (p = 0.03; H/L ratio = 3.50), PTHLH (p = 0.03; H/L ratio = 3.69), MMP8 (p = 0.01; H/L ratio =4.41) and SCNN1G (p = 0.04; H/L ratio = 3.42). Although selected miRNAs showed similar expression levels between H and L groups, significant correlation was found between the expression level of ssc-miR-133a (p < 0.01) and ssc-miR-92a (p < 0.01) and validated genes. These results provide a better understanding of the genetic architecture of prolificacy-related traits and embryo implantation failure in pigs.

Development of a baculovirus vector carrying a small hairpin RNA for suppression of sf-caspase-1 expression and improvement of recombinant protein production.

PubMed

Zhang, Xiaoyue; Xu, Keyan; Ou, Yanmei; Xu, Xiaodong; Chen, Hongying

2018-05-02

The Baculovirus expression vector system (BEVS) is a transient expression platform for recombinant protein production in insect cells. Baculovirus infection of insect cells will shutoff host translation and induce apoptosis and lead to the termination of protein expression. Previous reports have demonstrated the enhancement of protein yield in BEVS using stable insect cell lines expressing interference RNA to suppress the expression of caspase-1. In this study, short-hairpin RNA (shRNA) expression cassettes targeting Spodoptera frugiperda caspase-1 (Sf-caspase-1) were constructed and inserted into an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) vector. Using the recombinant baculovirus vectors, we detected the suppression of Sf-caspase-1 expression and cell apoptosis. Green fluorescent protein (GFP), Discosoma sp. Red (DsRed) and firefly luciferase were then expressed as reporter proteins. The results showed that suppression of apoptosis enhanced the accumulation of exogenous proteins at 2 and 3 days post infection. After 4 days post infection, the activity of the reporter proteins remained higher in BEVS using the baculovirus carrying shRNA in comparison with the control without shRNA, but the accumulated protein levels showed no obvious difference between them, suggesting that apoptosis suppression resulted in improved protein folding rather than translation efficiency at the very late stage of baculovirus infection. The baculovirus vector developed in this study would be a useful tool for the production of active proteins suitable for structural and functional studies or pharmaceutical applications in Sf9 cells, and it also has the potential to be adapted for the improvement of protein expression in different insect cell lines that can be infected by AcMNPV.

Heterogeneity in Immune Marker Expression after Acquisition of Resistance to EGFR Kinase Inhibitors: Analysis of a Case with Small Cell Lung Cancer Transformation.

PubMed

Suda, Kenichi; Murakami, Isao; Yu, Hui; Kim, Jihye; Ellison, Kim; Rivard, Christopher J; Mitsudomi, Tetsuya; Hirsch, Fred R

2017-06-01

Expression of immune markers is of scientific interest because of their potential roles as predictive biomarkers for immunotherapy. Although the microenvironment of metastatic tumors and/or therapy-inducible histological transformation may affect the expression of these immune markers, there are few data regarding this context. A 76-year-old never-smoking female with EGFR-mutated lung adenocarcinoma (AC) acquired resistance to gefitinib. After her death, an autopsy revealed SCLC transformation and EGFR T790M secondary mutation (T790M) as mutually exclusive resistance mechanisms occurring differently in different metastases; two liver metastases (SCLC versus AC with T790M) and two lymph node metastases (SCLC versus AC with T790M) were analyzed to compare the expression status of immune markers by immunohistochemistry and by an immune oncology gene expression panel. Programmed death ligand 1 (PD-L1) protein was partially expressed in tumor cells with AC lesions (T790M) but not in tumor cells with SCLC transformation. The liver metastasis with SCLC transformation showed no stromal PD-L1 expression and scant tumor-infiltrating lymphocytes, whereas the other lesions demonstrated stromal PD-L1 staining and infiltration of CD8-positive T cells. Data generated using an immuno-oncology gene expression panel indicated a higher level of T-cell costimulatory molecules and lower expression of type I interferon-regulated genes in lesions with SCLC transformation. These data highlight the heterogeneity of expression of immune markers depending on the metastatic sites and histological transformation and indicate that the biopsy specimen from one lesion may not be representative of immune marker status for all lesions. Copyright © 2017 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Mouse mammary tumour virus (MMTV) and human breast cancer with neuroendocrine differentiation.

PubMed

Js, Lawson; Cc, Ngan; Wk, Glenn; Dd, Tran

2017-01-01

Mouse mammary tumour viruses (MMTVs) may have a role in a subset of human breast cancers. MMTV positive human breast cancers have similar histological characteristics to neuroendocrine breast cancers and to MMTV positive mouse mammary tumours. The purpose of this study was to investigate the expression of neuroendocrine biomarkers - synaptophysin and chromogranin, to determine if these histological characteristics and biomarker expression were due to the influences of MMTV. Immunohistochemistry analyses to identify synaptophysin and chromogranin were conducted on a series of human breast cancers in which (i) MMTV had been previously identified and had similar histological characteristics to MMTV positive mouse mammary tumours and (ii) MMTV positive mouse mammary tumours. The expression of synaptophysin and chromogranin in MMTV positive mouse mammary tumors were all positive (7 of 7 specimens - 100% positive). The expression of synaptophysin and chromogranin in MMTV positive human breast cancers was much less prevalent (3 of 22 - 14%). There was no expression of synaptophysin and chromogranin in the normal breast tissue control specimens. It is not possible to draw any firm conclusions from these observations. However, despite the small numbers of MMTV positive mouse mammary tumours in this study, the universal expression in these specimens of synaptophysin and chromogranin proteins is striking. This pattern of synaptophysin and chromogranin expression is very different from their expression in MMTV positive human breast cancers. The reason for these differences is not known. The high prevalence of positive expression of synaptophysin and chromogranin in MMTV positive mouse mammary tumours and low expression of synaptophysin and chromogranin in MMTV positive human breast cancers indicates that MMTV is not usually associated with neuroendocrine human breast cancers.