Transcriptome and Degradome Sequencing Reveals Dormancy Mechanisms of Cunninghamia lanceolata Seeds1

PubMed Central

Xu, Huimin; Liu, Yongxiu; Soppe, Wim J.J.; Lin, Jinxing

2016-01-01

Seeds with physiological dormancy usually experience primary and secondary dormancy in the nature; however, little is known about the differential regulation of primary and secondary dormancy. We combined multiple approaches to investigate cytological changes, hormonal levels, and gene expression dynamics in Cunninghamia lanceolata seeds during primary dormancy release and secondary dormancy induction. Light microscopy and transmission electron microscopy revealed that protein bodies in the embryo cells coalesced during primary dormancy release and then separated during secondary dormancy induction. Transcriptomic profiling demonstrated that expression of genes negatively regulating gibberellic acid (GA) sensitivity reduced specifically during primary dormancy release, whereas the expression of genes positively regulating abscisic acid (ABA) biosynthesis increased during secondary dormancy induction. Parallel analysis of RNA ends revealed uncapped transcripts for ∼55% of all unigenes. A negative correlation between fold changes in expression levels of uncapped versus capped mRNAs was observed during primary dormancy release. However, this correlation was loose during secondary dormancy induction. Our analyses suggest that the reversible changes in cytology and gene expression during dormancy release and induction are related to ABA/GA balance. Moreover, mRNA degradation functions as a critical posttranscriptional regulator during primary dormancy release. These findings provide a mechanistic framework for understanding physiological dormancy in seeds. PMID:27760880

Liver cell-derived microparticles activate hedgehog signaling and alter gene expression in hepatic endothelial cells.

PubMed

Witek, Rafal P; Yang, Liu; Liu, Renshui; Jung, Youngmi; Omenetti, Alessia; Syn, Wing-Kin; Choi, Steve S; Cheong, Yeiwon; Fearing, Caitlin M; Agboola, Kolade M; Chen, Wei; Diehl, Anna Mae

2009-01-01

Angiogenesis contributes to vascular remodeling during cirrhosis. In cirrhotic livers, cholangiocytes, and myofibroblastic hepatic stellate cells (MF-HSC) produce Hedgehog (Hh) ligands. During embryogenesis Hh ligands are released from ligand-producing cells in microparticles and activate Hh signaling in endothelial cells. We studied whether adult liver cell-derived microparticles contain Hh ligands that alter hepatic sinusoidal endothelial cells (SEC). MF-HSC and cholangiocytes were exposed to platelet-derived growth factor to induce Hh ligands; microparticles were isolated from medium, analyzed by transmission electron microscopy and immunoblots, and applied to Hh-reporter-containing cells. Microparticles were obtained from serum and bile of rats after bile duct ligation (BDL) or sham surgery and applied to normal primary liver SEC with or without cyclopamine, an Hh signaling inhibitor. Effects on SEC gene expression were evaluated by quantitative reverse-transcription polymerase chain reaction and immunoblotting. Hh target gene expression and SEC activation markers were compared in primary SEC and in liver sections from healthy and BDL rats. Platelet-derived growth factor-treated MF-HSC and cholangiocytes released exosome-enriched microparticles containing biologically-active Hh ligands. BDL increased release of Hh-containing exosome-enriched microparticles into plasma and bile. Transmission electron microscopy and immunoblots revealed similarities among microparticles from all sources; all microparticles induced similar Hh-dependent changes in SEC gene expression. SEC from healthy livers did not express Hh target genes or activation markers, but both were up-regulated in SEC after BDL. Hh-containing exosome-enriched microparticles released from liver cells alter hepatic SEC gene expression, suggesting a novel mechanism for cirrhotic vasculopathy.

Detecting cells in time varying intensity images in confocal microscopy for gene expression studies in living cells

NASA Astrophysics Data System (ADS)

Mitra, Debasis; Boutchko, Rostyslav; Ray, Judhajeet; Nilsen-Hamilton, Marit

2015-03-01

In this work we present a time-lapsed confocal microscopy image analysis technique for an automated gene expression study of multiple single living cells. Fluorescence Resonance Energy Transfer (FRET) is a technology by which molecule-to-molecule interactions are visualized. We analyzed a dynamic series of ~102 images obtained using confocal microscopy of fluorescence in yeast cells containing RNA reporters that give a FRET signal when the gene promoter is activated. For each time frame, separate images are available for three spectral channels and the integrated intensity snapshot of the system. A large number of time-lapsed frames must be analyzed to identify each cell individually across time and space, as it is moving in and out of the focal plane of the microscope. This makes it a difficult image processing problem. We have proposed an algorithm here, based on scale-space technique, which solves the problem satisfactorily. The algorithm has multiple directions for even further improvement. The ability to rapidly measure changes in gene expression simultaneously in many cells in a population will open the opportunity for real-time studies of the heterogeneity of genetic response in a living cell population and the interactions between cells that occur in a mixed population, such as the ones found in the organs and tissues of multicellular organisms.

In vitro Paracoccidioides brasiliensis biofilm and gene expression of adhesins and hydrolytic enzymes.

PubMed

Sardi, Janaina de Cássia Orlandi; Pitangui, Nayla de Souza; Voltan, Aline Raquel; Braz, Jaqueline Derissi; Machado, Marcelo Pelajo; Fusco Almeida, Ana Marisa; Mendes Giannini, Maria Jose Soares

2015-01-01

Paracoccidioides species are dimorphic fungi that initially infect the lungs but can also spread throughout the body. The spreading infection is most likely due to the formation of a biofilm that makes it difficult for the host to eliminate the infection. Biofilm formation is crucial for the development of infections and confines the pathogen to an extracellular matrix. Its presence is associated with antimicrobial resistance and avoidance of host defenses. This current study provides the first description of biofilm formation by Paracoccidioides brasiliensis (Pb18) and an analysis of gene expression, using real-time PCR, associated with 3 adhesins and 2 hydrolytic enzymes that could be associated with the virulence profile. Biofilm formation was analyzed using fluorescence microscopy, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Metabolic activity was determined using the XTT reduction assay. P. brasiliensis was able to form mature biofilm in 144 h with a thickness of 100 μm. The presence of a biofilm was found to be associated with an increase in the expression of adhesins and enzymes. GP43, enolase, GAPDH and aspartyl proteinase genes were over-expressed, whereas phospholipase was down-regulated in biofilm. The characterization of biofilm formed by P. brasiliensis may contribute to a better understanding of the pathogenesis of paracoccidioidomycosis as well as the search for new therapeutic alternatives; while improving the effectiveness of treatment.

Spatiotemporal evolution of bacterial biofilm colonies

NASA Astrophysics Data System (ADS)

Wilking, James; Koehler, Stephan; Sinha, Naveen; Seminara, Agnese; Brenner, Michael; Weitz, David

2014-03-01

Many bacteria on earth live in surface-attached communities known as biofilms. Gene expression in a biofilm is typically varied, resulting in a variety of phenotypes within a single film. These phenotypes play a critical role in biofilm physiology and development. We use time-resolved, wide-field fluorescence microscopy to image triple-labeled fluorescent Bacillus Subtilis colonies grown on agar to determine in a non-invasive fashion the evolving phenotypes. We infer their transition rates from the resulting spatiotemporal maps of gene expression. Moreover, we correlate these transition rates with local measurements of nutrient concentration to determine the influence of extracellular signals on gene expression.

Ovarian Tumor-Stroma Interactions in an In Vivo Orthotopic Model

DTIC Science & Technology

2011-08-01

cancer cells to the novel environment. We have devised an Intravital Video Microscopy approach to this problem in which MOVCAR cells labeled with green...Ovarian cancer, gene expression, metastasis, intravital video microscopy 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER...placed in a dorsal skin-fold chamber for Intravital Video Microscopy (IVM). The minced pseudo-organ tissue revascularizes and recapitulates some of the

A high-throughput quantitative expression analysis of cancer-related genes in human HepG2 cells in response to limonene, a potential anticancer agent.

PubMed

Hafidh, Rand R; Hussein, Saba Z; MalAllah, Mohammed Q; Abdulamir, Ahmed S; Abu Bakar, Fatimah

2017-11-14

Citrus bioactive compounds, as active anticancer agent, have been under focus by several studies worldwide. However, the underlying genes responsible for the anticancer potential have not been sufficiently highlighted. The current study investigated the gene expression profile of hepatocellular carcinoma, HepG2, cells after treatment with Limonene. The concentration that killed 50% of HepG2 cells was used to elucidate the genetic mechanisms of limonene anticancer activity. The apoptotic induction was detected by flow cytometry and confocal fluorescence microscope. Two of pro-apoptotic events, caspase-3 activation and phosphatidylserine translocation were manifested by confocal fluorescence microscopy. High-throughput real-time PCR was used to profile 1023 cancer-related genes in 16 different gene families related to the cancer development. In comparison to untreated cells, limonene increased the percentage of apoptotic cells up to 89.61%, by flow cytometry, and 48.2% by fluorescence microscopy. There was a significant limonene-driven differential gene expression of HepG2 cells in 15 different gene families. Limonene was shown to significantly (>2log) up-regulate and down-regulate 14 and 59 genes, respectively. The affected gene families, from most to least affected, were apoptosis induction, signal transduction, cancer genes augmentation, alteration in kinases expression, inflammation, DNA damage repair, and cell cycle proteins. The current study reveals that limonene could be a promising, cheap, and effective anticancer compound. The broad spectrum of limonene anticancer activity is interesting for anticancer drug development. Further research is needed to confirm the current findings and to examine the anticancer potential of limonene along with underlying mechanisms on different cell lines. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Changes in gravitational force induce alterations in gene expression that can be monitored in the live, developing zebrafish heart

NASA Astrophysics Data System (ADS)

Gillette-Ferguson, I.; Ferguson, D. G.; Poss, K. D.; Moorman, S. J.

2003-10-01

Little is known about the effect of microgravity on gene expression, particularly in vivo during embryonic development. Using transgenic zebrafish that express the gfp gene under the influence of a β-actin promoter, we examined the affect of simulated-microgravity on GFP expression in the heart. Zebrafish embryos, at the 18-20 somite-stage, were exposed to simulated-microgravity for 24 hours. The intensity of GFP fluorescence associated with the heart was then determined using fluorescence microscopy. Our measurements indicated that simulated-microgravity induced a 23.9% increase in GFP-associated fluorescence in the heart. In contrast, the caudal notochord showed a 17.5% increase and the embryo as a whole showed only an 8.5% increase in GFP-associated fluorescence. This suggests that there are specific effects on the heart causing the more dramatic increase. These studies indicate that microgravity can influence gene expression and demonstrate the usefulness of this in vivo model of "reporter-gene" expression for studying the effects of microgravity.

Expression of the core antigen gene of hepatitis B virus (HBV) in Acetobacter methanolicus using broad-host-range vectors.

PubMed

Schröder, R; Maassen, A; Lippoldt, A; Börner, T; von Baehr, R; Dobrowolski, P

1991-08-01

Using the broad-host-range promoter probe vector pRS201 for cloning of phage Acm1 promoters, we established a convenient vector system for expression of heterologous genes in different Gram-negative bacteria. The usefulness of this system was demonstrated by expression of the HBV core gene in Acetobacter methanolicus. Plasmids carrying the HBV core gene downstream of different Acm1-phage promoters were transferred to A. methanolicus, a new potential host for recombinant DNA expression. Using enzyme immunoassay and immunoblot techniques, the amount and composition of core antigen produced in A. methanolicus were compared with that derived from Escherichia coli. The expression of immunoreactive core antigen in A. methanolicus exceeds by sevenfold that in E. coli using an expression system with tandemly arranged promoters. Morphological observations by electron microscopy show that the HBV core gene products isolated from both hosts are assembled into regular spherical particles with a diameter of about 28 nm that are comparable to original viral nucleocapsids.

Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues

PubMed Central

Lee, Je Hyuk; Daugharthy, Evan R.; Scheiman, Jonathan; Kalhor, Reza; Ferrante, Thomas C.; Terry, Richard; Turczyk, Brian M.; Yang, Joyce L.; Lee, Ho Suk; Aach, John; Zhang, Kun; Church, George M.

2014-01-01

RNA sequencing measures the quantitative change in gene expression over the whole transcriptome, but it lacks spatial context. On the other hand, in situ hybridization provides the location of gene expression, but only for a small number of genes. Here we detail a protocol for genome-wide profiling of gene expression in situ in fixed cells and tissues, in which RNA is converted into cross-linked cDNA amplicons and sequenced manually on a confocal microscope. Unlike traditional RNA-seq our method enriches for context-specific transcripts over house-keeping and/or structural RNA, and it preserves the tissue architecture for RNA localization studies. Our protocol is written for researchers experienced in cell microscopy with minimal computing skills. Library construction and sequencing can be completed within 14 d, with image analysis requiring an additional 2 d. PMID:25675209

The formation of the light-sensing compartment of cone photoreceptors coincides with a transcriptional switch

PubMed Central

Daum, Janine M; Keles, Özkan; Holwerda, Sjoerd JB; Kohler, Hubertus; Rijli, Filippo M

2017-01-01

High-resolution daylight vision is mediated by cone photoreceptors. The molecular program responsible for the formation of their light sensor, the outer segment, is not well understood. We correlated daily changes in ultrastructure and gene expression in postmitotic mouse cones, between birth and eye opening, using serial block-face electron microscopy (EM) and RNA sequencing. Outer segments appeared rapidly at postnatal day six and their appearance coincided with a switch in gene expression. The switch affected over 14% of all expressed genes. Genes that switched off were rich in transcription factors and neurogenic genes. Those that switched on contained genes relevant for cone function. Chromatin rearrangements in enhancer regions occurred before the switch was completed, but not after. We provide a resource comprised of correlated EM, RNAseq, and ATACseq data, showing that the growth of a key compartment of a postmitotic cell involves an extensive switch in gene expression and chromatin accessibility. PMID:29106373

Emodin affects biofilm formation and expression of virulence factors in Streptococcus suis ATCC700794.

PubMed

Yang, Yan-Bei; Wang, Shuai; Wang, Chang; Huang, Quan-Yong; Bai, Jing-Wen; Chen, Jian-Qing; Chen, Xue-Ying; Li, Yan-Hua

2015-12-01

Streptococcus suis (S. suis) is a swine pathogen and also a zoonotic agent. In this study, the effects of subinhibitory concentrations (sub-MICs) of emodin on biofilm formation by S. suis ATCC700794 were evaluated. As quantified by crystal violet staining, biofilm formation by S. suis ATCC700794 was dose-dependently decreased after growth with 1/2 MIC, 1/4 MIC, or 1/8 MIC of emodin. By scanning electron microscopy, the structural architecture of the S. suis ATCC700794 biofilms was examined following growth in culture medium supplemented with 1/2 MIC, 1/4 MIC, 1/8 MIC, or 1/16 MIC of emodin. Scanning electron microscopy analysis revealed the potential effect of emodin on biofilm formation by S. suis ATCC700794. The expression of luxS gene and virulence genes in S. suis ATCC700794 was investigated by quantitative RT-PCR. It was found that sub-MICs of emodin significantly decreased the expression of gapdh, sly, fbps, ef, and luxS. However, it was found that sub-MICs of emodin significantly increased the expression of cps2J, mrp, and gdh. These findings showed that sub-MICs of emodin could cause the difference in the expression level of the virulence genes.

Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy

PubMed Central

Mazo-Vargas, Anyimilehidi; Park, Heungwon; Aydin, Mert; Buchler, Nicolas E.

2014-01-01

Time-lapse fluorescence microscopy is an important tool for measuring in vivo gene dynamics in single cells. However, fluorescent proteins are limited by slow chromophore maturation times and the cellular autofluorescence or phototoxicity that arises from light excitation. An alternative is luciferase, an enzyme that emits photons and is active upon folding. The photon flux per luciferase is significantly lower than that for fluorescent proteins. Thus time-lapse luminescence microscopy has been successfully used to track gene dynamics only in larger organisms and for slower processes, for which more total photons can be collected in one exposure. Here we tested green, yellow, and red beetle luciferases and optimized substrate conditions for in vivo luminescence. By combining time-lapse luminescence microscopy with a microfluidic device, we tracked the dynamics of cell cycle genes in single yeast with subminute exposure times over many generations. Our method was faster and in cells with much smaller volumes than previous work. Fluorescence of an optimized reporter (Venus) lagged luminescence by 15–20 min, which is consistent with its known rate of chromophore maturation in yeast. Our work demonstrates that luciferases are better than fluorescent proteins at faithfully tracking the underlying gene expression. PMID:25232010

Immune gene expression for diverse haemocytes derived from pacific white shrimp, Litopenaeus vannamei.

PubMed

Yang, Chih-Chiu; Lu, Chung-Lun; Chen, Sherwin; Liao, Wen-Liang; Chen, Shiu-Nan

2015-05-01

In this study, diverse haemocytes from Pacific white shrimp Litopenaeus vannamei were spread by flow cytometer sorting system. Using the two commonly flow cytometric parameters FSC and SSC, the haemocytes could be divided into three populations. Microscopy observation of L. vannamei haemocytes in anticoagulant buffer revealed three morphologically distinct cell types designated as granular cell, hyaline cell and semigranular cell. Immune genes, which includes prophenoloxidase (proPO), lipopolysaccharide-β-glucan binding protein (LGBP), peroxinectin, crustin, lysozyme, penaeid-3a and transglutaminase (TGase), expressed from different haemocyte were analysed by quantitative real time PCR (qPCR). Results from the mRNA expression was estimated by relative level of each gene to β-actin gene. Finally, the seven genes could be grouped by their dominant expression sites. ProPO, LGBP and peroxinectin were highly expressed in granular cells, while LGBP, crustin, lysozyme and P-3a were highly expressed in semigranular cells and TGase was highly expressed in hyaline cells. In this study, L. vannamei haemocytes were firstly grouped into three different types and the immune related genes expression in grouped haemocytes were estimated. Copyright © 2015 Elsevier Ltd. All rights reserved.

Optimisations and Challenges Involved in the Creation of Various Bioluminescent and Fluorescent Influenza A Virus Strains for In Vitro and In Vivo Applications

PubMed Central

Herfst, Sander; Bestebroer, Theo M.; Vaes, Vincent P.; van der Hoeven, Barbara; Koster, Abraham J.; Kremers, Gert-Jan; Scott, Dana P.; Gultyaev, Alexander P.; Sorell, Erin M.; de Graaf, Miranda; Bárcena, Montserrat; Rimmelzwaan, Guus F.; Fouchier, Ron A.

2015-01-01

Bioluminescent and fluorescent influenza A viruses offer new opportunities to study influenza virus replication, tropism and pathogenesis. To date, several influenza A reporter viruses have been described. These strategies typically focused on a single reporter gene (either bioluminescent or fluorescent) in a single virus backbone. However, whilst bioluminescence is suited to in vivo imaging, fluorescent viruses are more appropriate for microscopy. Therefore, the idea l reporter virus varies depending on the experiment in question, and it is important that any reporter virus strategy can be adapted accordingly. Herein, a strategy was developed to create five different reporter viruses in a single virus backbone. Specifically, enhanced green fluorescent protein (eGFP), far-red fluorescent protein (fRFP), near-infrared fluorescent protein (iRFP), Gaussia luciferase (gLUC) and firefly luciferase (fLUC) were inserted into the PA gene segment of A/PR/8/34 (H1N1). This study provides a comprehensive characterisation of the effects of different reporter genes on influenza virus replication and reporter activity. In vivo reporter gene expression, in lung tissues, was only detected for eGFP, fRFP and gLUC expressing viruses. In vitro, the eGFP-expressing virus displayed the best reporter stability and could be used for correlative light electron microscopy (CLEM). This strategy was then used to create eGFP-expressing viruses consisting entirely of pandemic H1N1, highly pathogenic avian influenza (HPAI) H5N1 and H7N9. The HPAI H5N1 eGFP-expressing virus infected mice and reporter gene expression was detected, in lung tissues, in vivo. Thus, this study provides new tools and insights for the creation of bioluminescent and fluorescent influenza A reporter viruses. PMID:26241861

Isolation of Optically Targeted Single Bacteria by Application of Fluidic Force Microscopy to Aerobic Anoxygenic Phototrophs from the Phyllosphere

PubMed Central

Stiefel, Philipp; Zambelli, Tomaso

2013-01-01

In their natural environment, bacteria often behave differently than they do under laboratory conditions. To gain insight into the physiology of bacteria in situ, dedicated approaches are required to monitor their adaptations and specific behaviors under environmental conditions. Optical microscopy is crucial for the observation of fundamental characteristics of bacteria, such as cell shape, size, and marker gene expression. Here, fluidic force microscopy (FluidFM) was exploited to isolate optically selected bacteria for subsequent identification and characterization. In this study, bacteriochlorophyll-producing bacteria, which can be visualized due to their characteristic fluorescence in the infrared range, were isolated from leaf washes. Bacterial communities from the phyllosphere were investigated because they harbor genes indicative of aerobic anoxygenic photosynthesis. Our data show that different species of Methylobacterium express their photosystem in planta, and they show a distinct pattern of bacteriochlorophyll production under laboratory conditions that is dependent on supplied carbon sources. PMID:23770907

Heterogeneous gene expression and functional activity of ryanodine receptors in resistance and conduit pulmonary as well as mesenteric artery smooth muscle cells.

PubMed

Zheng, Yun-Min; Wang, Qing-Song; Liu, Qing-Hua; Rathore, Rakesh; Yadav, Vishal; Wang, Yong-Xiao

2008-01-01

Hypoxia causes heterogeneous contractile responses in resistance and conduit pulmonary as well as systemic (mesenteric) artery smooth muscle cells (RPASMCs, CPASMCs and MASMCs), but the underlying mechanisms are largely unknown. In this study, we aimed to investigate whether the gene expression and functional activity of ryanodine receptors (RyRs) would be different in these 3 cell types. RyR mRNA expression, Ca(2+) sparks and [Ca(2+)](i) were measured by real-time quantitative RT-PCR, laser scanning confocal microscopy and wide-field fluorescence microscopy, respectively. All 3 RyR subtype (RyR1, RyR2 and RyR3) mRNAs are expressed in RPASMCs, CPASMCs and MASMCs, but their expression levels are different. Spontaneous Ca(2+) sparks (functional events of RyRs) show distinct frequency, amplitude, duration, size and kinetics in these 3 cell types. Similarly, activation of RyRs by caffeine, 4-chloro-m-cresol or high K(+) induces differential Ca(2+) release. Moreover, hypoxia-induced increase in [Ca(2+)](i) is largest in MASMCs relative to CPSAMCs and smallest in RPASMCs. This study provides comprehensive evidence that RyRs are heterogeneous in gene expression and functional activity in RPASMCs, CPASMCs and MASMCs, which may contribute to the diversity of excitation-contraction coupling and hypoxic Ca(2+) responses in different vascular smooth muscle cells. Copyright 2008 S. Karger AG, Basel.

Superparamagnetic iron oxide nanoparticle-labeled cells as an effective vehicle for tracking the GFP gene marker using magnetic resonance imaging

PubMed Central

Zhang, Z; Mascheri, N; Dharmakumar, R; Fan, Z; Paunesku, T; Woloschak, G; Li, D

2010-01-01

Background Detection of a gene using magnetic resonance imaging (MRI) is hindered by the magnetic resonance (MR) targeting gene technique. Therefore it may be advantageous to image gene-expressing cells labeled with superparamagnetic iron oxide (SPIO) nanoparticles by MRI. Methods The GFP-R3230Ac (GFP) cell line was incubated for 24 h using SPIO nanoparticles at a concentration of 20 μg Fe/mL. Cell samples were prepared for iron content analysis and cell function evaluation. The labeled cells were imaged using fluorescent microscopy and MRI. Results SPIO was used to label GFP cells effectively, with no effects on cell function and GFP expression. Iron-loaded GFP cells were successfully imaged with both fluorescent microscopy and T2*-weighted MRI. Prussian blue staining showed intracellular iron accumulation in the cells. All cells were labeled (100% labeling efficiency). The average iron content per cell was 4.75±0.11 pg Fe/cell (P<0.05 versus control). Discussion This study demonstrates that the GFP expression of cells is not altered by the SPIO labeling process. SPIO-labeled GFP cells can be visualized by MRI; therefore, GFP, a gene marker, was tracked indirectly with the SPIO-loaded cells using MRI. The technique holds promise for monitoring the temporal and spatial migration of cells with a gene marker and enhancing the understanding of cell- and gene-based therapeutic strategies. PMID:18956269

GENE EXPRESSION CHANGES AFTER SEIZURE PRECONDITIONING IN THE THREE MAJOR HIPPOCAMPAL CELL LAYERS

PubMed Central

Borges, Karin; Shaw, Renee; Dingledine, Raymond

2008-01-01

Rodents experience hippocampal damage after status epilepticus (SE) mainly in pyramidal cells while sparing the dentate granule cell layer (DGCL). Hippocampal damage was prevented in rats that had been preconditioned by brief seizures on two consecutive days before SE. To identify neuroprotective genes and biochemical pathways changed after preconditioning we compared the effect of preconditioning on gene expression in the CA1 and CA3 pyramidal and DGCLs, harvested by laser capture microscopy. In the DGCL the expression of 632 genes was altered, compared to only 151 and 58 genes in CA1 and CA3 pyramidal cell layers. Most of the differentially expressed genes regulate tissue structure and intra- and extracellular signaling, including neurotransmission. A selective upregulation of energy metabolism transcripts occurred in CA1 pyramidal cells relative to the DGCL. These results reveal a broad transcriptional response of the DGCL to preconditioning, and suggest several mechanisms underlying the neuroprotective effect of preconditioning seizures. PMID:17239605

TM6SF2 is a regulator of liver fat metabolism influencing triglyceride secretion and hepatic lipid droplet content

PubMed Central

Mahdessian, Hovsep; Taxiarchis, Apostolos; Popov, Sergej; Silveira, Angela; Franco-Cereceda, Anders; Hamsten, Anders; Eriksson, Per; van't Hooft, Ferdinand

2014-01-01

Genome-wide association studies have identified a locus on chromosome 19 associated with plasma triglyceride (TG) concentration and nonalcoholic fatty liver disease. However, the identity and functional role of the gene(s) responsible for these associations remain unknown. Of 19 expressed genes contained in this locus, none has previously been implicated in lipid metabolism. We performed gene expression studies and expression quantitative trait locus analysis in 206 human liver samples to identify the putative causal gene. Transmembrane 6 superfamily member 2 (TM6SF2), a gene with hitherto unknown function, expressed predominantly in liver and intestine, was identified as the putative causal gene. TM6SF2 encodes a protein of 351 amino acids with 7–10 predicted transmembrane domains. Otherwise, no other protein features were identified which could help to elucidate the function of TM6SF2. Protein subcellular localization studies with confocal microscopy demonstrated that TM6SF2 is localized in the endoplasmic reticulum and the ER-Golgi intermediate compartment of human liver cells. Functional studies for secretion of TG-rich lipoproteins (TRLs) and lipid droplet content were performed in human hepatoma Huh7 and HepG2 cells using confocal microscopy and siRNA inhibition and overexpression techniques. In agreement with the genome-wide association data, it was found that TM6SF2 siRNA inhibition was associated with reduced secretion of TRLs and increased cellular TG concentration and lipid droplet content, whereas TM6SF2 overexpression reduced liver cell steatosis. We conclude that TM6SF2 is a regulator of liver fat metabolism with opposing effects on the secretion of TRLs and hepatic lipid droplet content. PMID:24927523

Radiofrequency-enhanced vascular gene transduction and expression for intravascular MR imaging-guided therapy: feasibility study in pigs.

PubMed

Du, Xiangying; Qiu, Bensheng; Zhan, Xiangcan; Kolmakova, Antonina; Gao, Fabao; Hofmann, Lawrence V; Cheng, Linzhao; Chatterjee, Subroto; Yang, Xiaoming

2005-09-01

To evaluate the feasibility of radiofrequency (RF)-enhanced vascular gene transduction and expression by using a magnetic resonance (MR) imaging-heating guidewire as an intravascular heating vehicle during MR imaging-guided therapy. The institutional committee for animal care and use approved the experimental protocol. The study included in vitro evaluation of the use of RF energy to enhance gene transduction and expression in vascular cells, as well as in vivo validation of the feasibility of intravascular MR imaging-guided RF-enhanced vascular gene transduction and expression in pig arteries. For in vitro experiments, approximately 10(4) vascular smooth muscle cells were seeded in each of four chambers of a cell culture plate. Next, 1 mL of a green fluorescent protein gene (gfp)-bearing lentivirus was added to each chamber. Chamber 4 was heated at approximately 41 degrees C for 15 minutes by using an MR imaging-heating guidewire connected to a custom RF generator. At day 6 after transduction, the four chambers were examined and compared at confocal microscopy to determine the efficiency of gfp transduction and expression. For the in vivo experiments, a lentivirus vector bearing a therapeutic gene, vascular endothelial growth factor 165 (VEGF-165), was transferred by using a gene delivery balloon catheter in 18 femoral-iliac arteries (nine artery pairs) in domestic pigs and Yucatan pigs with atherosclerosis. During gene infusion, one femoral-iliac artery in each pig was heated to approximately 41 degrees C with RF energy transferred via the intravascular MR imaging-heating guidewire, while the contralateral artery was not heated (control condition). At day 6, the 18 arteries were harvested for quantitative Western blot analysis to compare VEGF-165 transduction and expression efficiency between RF-heated and nonheated arterial groups. Confocal microscopy showed gfp expression in chamber 4 that was 293% the level of expression in chamber 1 (49.6% +/- 25.8 vs 16.8% +/- 8.0). Results of Western blot analysis showed VEGF-165 expression for normal arteries in the RF-heated group that was 300% the level of expression in the nonheated group (70.4 arbitrary units [au] +/- 107.1 vs 23.5 au +/- 29.8), and, for atherosclerotic arteries in the RF-heated group, 986% the level in the nonheated group (129.2 au +/- 100.3 vs 13.1 au +/- 4.9). Simultaneous monitoring and enhancement of vascular gene delivery and expression is feasible with the MR imaging-heating guidewire.

Strain Library Imaging Protocol for high-throughput, automated single-cell microscopy of large bacterial collections arrayed on multiwell plates.

PubMed

Shi, Handuo; Colavin, Alexandre; Lee, Timothy K; Huang, Kerwyn Casey

2017-02-01

Single-cell microscopy is a powerful tool for studying gene functions using strain libraries, but it suffers from throughput limitations. Here we describe the Strain Library Imaging Protocol (SLIP), which is a high-throughput, automated microscopy workflow for large strain collections that requires minimal user involvement. SLIP involves transferring arrayed bacterial cultures from multiwell plates onto large agar pads using inexpensive replicator pins and automatically imaging the resulting single cells. The acquired images are subsequently reviewed and analyzed by custom MATLAB scripts that segment single-cell contours and extract quantitative metrics. SLIP yields rich data sets on cell morphology and gene expression that illustrate the function of certain genes and the connections among strains in a library. For a library arrayed on 96-well plates, image acquisition can be completed within 4 min per plate.

Evaluation of Magnetic Nanoparticle-Labeled Chondrocytes Cultivated on a Type II Collagen–Chitosan/Poly(Lactic-co-Glycolic) Acid Biphasic Scaffold

PubMed Central

Su, Juin-Yih; Chen, Shi-Hui; Chen, Yu-Pin; Chen, Wei-Chuan

2017-01-01

Chondral or osteochondral defects are still controversial problems in orthopedics. Here, chondrocytes labeled with magnetic nanoparticles were cultivated on a biphasic, type II collagen–chitosan/poly(lactic-co-glycolic acid) scaffold in an attempt to develop cultures with trackable cells exhibiting growth, differentiation, and regeneration. Rabbit chondrocytes were labeled with magnetic nanoparticles and characterized by scanning electron microscopy (SEM), transmission electron (TEM) microscopy, and gene and protein expression analyses. The experimental results showed that the magnetic nanoparticles did not affect the phenotype of chondrocytes after cell labeling, nor were protein and gene expression affected. The biphasic type II collagen–chitosan/poly(lactic-co-glycolic) acid scaffold was characterized by SEM, and labeled chondrocytes showed a homogeneous distribution throughout the scaffold after cultivation onto the polymer. Cellular phenotype remained unaltered but with increased gene expression of type II collagen and aggrecan, as indicated by cell staining, indicating chondrogenesis. Decreased SRY-related high mobility group-box gene (Sox-9) levels of cultured chondrocytes indicated that differentiation was associated with osteogenesis. These results are encouraging for the development of techniques for trackable cartilage regeneration and osteochondral defect repair which may be applied in vivo and, eventually, in clinical trials. PMID:28054960

[Subretinal transplantation of human fetal lung fibroblasts expressed ciliary neurotrophic factor gene prevent photoreceptor degeneration in RCS rats].

PubMed

Huang, Qian; Xu, Ping; Xia, Xin; Hu, Hong-hui; Wang, Feng; Li, Hui-ming

2006-02-01

To investigate the efficacy of subretinal transplantation of CNTF gene transfected fibroblasts for preventing photoreceptor degeneration in RCS. The human fetal lung fibroblasts with high level expression of CNTF were established by liposome mediated gene transfer and MTX selection. A 5 microl of cell suspension, containing 1 x 10(5) cells, was injected through pars plana of ciliary body into the subretinal space of the right eye at postnatal 4-5 weeks, the left eye was left without injection or injected with PBS as controls. The both eyes were enucleated for histopathological examinations at 2, 4, 6, 8, 10, 12 and 15 weeks following transplantation. The level of CNTF protein (91,046.15 pg/ml) expressed in the transfected cells was determined by sandwich enzyme-linked immunosorbent assay (ELISA). The four of seven eyes examined by light microscopy and the ten of 14 eyes examined by electro microscopy showed rescue effect. The prolonged photoreceptor survival, reduction of apoptotic cells and debris were observed in transplanted eyes in comparison with untreated or sham-injected eyes. This study provides the first indication that transplanted human fibroblasts with high level expression of CNTF are able to rescue photoreceptor degeneration in RCS dystrophic rat retina.

Adhesion, proliferation and differentiation of osteoblasts on zirconia films prepared by cathodic arc deposition.

PubMed

Zhang, Shailin; Sun, Junying; Xu, Ying; Qian, Shi; Wang, Bing; Liu, Fei; Liu, Xuanyong

2013-01-01

Zirconia films were prepared on titanium by cathodic arc deposition technique. The surface topography and element composition of the films were characterized by scanning electron microscopy and X-ray photoelectron spectroscopy, respectively. Osteoblast-like MG63 cells were cultured on the surface of the zirconia films in vitro, and cell behaviour was investigated, with titanium as control. The results obtained from scanning electron microscopy and immunofluorescence studies showed that the MG63 cells on ZrO2 films spread better than those on Ti. The CCK8 assay indicated that the zirconia films promoted the proliferation of MG63 cells. The results of alkaline phosphatase (ALP) activity test and the expression of osteogenic marker genes, such as ALP, collagen I and osteocalcin, demonstrated that the differentiation of MG63 cells might be enhanced by zirconia films. In addition, the zirconia films possibly regulated osteoclastogenic gene expression by stimulating the expression of osteoprotegerin and reducing the expression of receptor activator of nuclear factor-kappaB ligand. The present work suggests that the ZrO2 film is worth further consideration for orthopedic implant applications.

Early development of fern gametophytes in microgravity

NASA Technical Reports Server (NTRS)

Roux, Stanley J.; Chatterjee, Ani; Hillier, Sheila; Cannon, Tom

2003-01-01

Dormant spores of the fern Ceratopteris richardii were flown on Shuttle mission STS-93 to evaluate the effects of micro-g on their development and on their pattern of gene expression. Prior to flight the spores were sterilized and sown into one of two environments: (1) Microscope slides in a video-microscopy module; and (2) Petri dishes. All spores were then stored in darkness until use. Spore germination was initiated on orbit after exposure to light. For the spores on microscope slides, cell level changes were recorded through the clear spore coat of the spores by video microscopy. After their exposure to light, spores in petri dishes were frozen in orbit at four different time points during which on earth gravity fixes the polarity of their development. Spores were then stored frozen in Biological Research in Canister units until recovery on earth. The RNAs from these cells and from 1-g control cells were extracted and analyzed on earth after flight to assay changes in gene expression. Video microscopy results revealed that the germinated spores developed normally in microgravity, although the polarity of their development, which is guided by gravity on earth, was random in space. Differential Display-PCR analyses of RNA extracted from space-flown cells showed that there was about a 5% change in the pattern of gene expression between cells developing in micro-g compared to those developing on earth. c2002 Published by Elsevier Science Ltd on behalf of COSPAR.

Replication pattern of the pericentromeric region of chromosome 10q and expression of the RET protooncogene.

PubMed

Cinti, R; Schena, F; Passalacqua, M; Ceccherini, I; Ravazzolo, R

2004-08-15

Regulation of the RET gene is highly specific during embryo development and is strictly tissue-specific. Control of transcription depends on mechanisms influenced by epigenetic processes, in particular, histone acetylation at regions flanking the 5' end of the gene. Since the RET gene is mapped in the pericentromeric region of the human chromosome 10, the implication of epigenetic processes is even more striking and worth to be investigated in an extended chromosomal tract. One experimental approach to study the chromatin status in relationship with gene transcription is to assess the replication timing, which we did by using fluorescent in situ hybridization in cells expressing or not expressing the RET gene. By using probes spanning a 700-kb genomic region from the RET locus toward the centromere, we found a relationship between RET expression and early replication. Different patterns were observed between cells naturally expressing RET and cells induced to expression of RET by treatment with sodium butyrate, an inhibitor of histone deacetylases. Three-dimensional analysis of the nuclear localization of fluorescent signals by confocal microscopy showed difference of localization between the RET probe and a probe for a housekeeping gene, G3PDH, located at 12p13.3, in cells that do not express RET, in accordance with previous data for other genes and chromosomal regions. However, RET-expressing cells showed a localization of signals which was not consistent with that expected for expressed genes.

3D confocal reconstruction of gene expression in mouse.

PubMed

Hecksher-Sørensen, J; Sharpe, J

2001-01-01

Three-dimensional computer reconstructions of gene expression data will become a valuable tool in biomedical research in the near future. However, at present the process of converting in situ expression data into 3D models is a highly specialized and time-consuming procedure. Here we present a method which allows rapid reconstruction of whole-mount in situ data from mouse embryos. Mid-gestation embryos were stained with the alkaline phosphotase substrate Fast Red, which can be detected using confocal laser scanning microscopy (CLSM), and cut into 70 microm sections. Each section was then scanned and digitally reconstructed. Using this method it took two days to section, digitize and reconstruct the full expression pattern of Shh in an E9.5 embryo (a 3D model of this embryo can be seen at genex.hgu.mrc.ac.uk). Additionally we demonstrate that this technique allows gene expression to be studied at the single cell level in intact tissue.

Mitochondria-targeted antioxidants protect against amyloid-beta toxicity in Alzheimer's disease neurons.

PubMed

Manczak, Maria; Mao, Peizhong; Calkins, Marcus J; Cornea, Anda; Reddy, Arubala P; Murphy, Michael P; Szeto, Hazel H; Park, Byung; Reddy, P Hemachandra

2010-01-01

The purpose of our study was to investigate the effects of the mitochondria-targeted antioxidants, MitoQ and SS31, and the anti-aging agent resveratrol on neurons from a mouse model (Tg2576 line) of Alzheimer's disease (AD) and on mouse neuroblastoma (N2a) cells incubated with the amyloid-beta (Abeta) peptide. Using electron and confocal microscopy, gene expression analysis, and biochemical methods, we studied mitochondrial structure and function and neurite outgrowth in N2a cells treated with MitoQ, SS31, and resveratrol, and then incubated with Abeta. In N2a cells only incubated with the Abeta, we found increased expressions of mitochondrial fission genes and decreased expression of fusion genes and also decreased expression of peroxiredoxins. Electron microscopy of the N2a cells incubated with Abeta revealed a significantly increased number of mitochondria, indicating that Abeta fragments mitochondria. Biochemical analysis revealed that function is defective in mitochondria. Neurite outgrowth was significantly decreased in Abeta-incubated N2a cells, indicating that Abeta affects neurite outgrowth. However, in N2a cells treated with MitoQ, SS31, and resveratrol, and then incubated with Abeta, abnormal expression of peroxiredoxins and mitochondrial structural genes were prevented and mitochondrial function was normal; intact mitochondria were present and neurite outgrowth was significantly increased. In primary neurons from amyloid-beta precursor protein transgenic mice that were treated with MitoQ and SS31, neurite outgrowth was significantly increased and cyclophilin D expression was significantly decreased. These findings suggest that MitoQ and SS31 prevent Abeta toxicity, which would warrant the study of MitoQ and SS31 as potential drugs to treat patients with AD.

A plasmid-based Escherichia coli gene expression system with cell-to-cell variation below the extrinsic noise limit

PubMed Central

2017-01-01

Experiments in synthetic biology and microbiology can benefit from protein expression systems with low cell-to-cell variability (noise) and expression levels precisely tunable across a useful dynamic range. Despite advances in understanding the molecular biology of microbial gene regulation, many experiments employ protein-expression systems exhibiting high noise and nearly all-or-none responses to induction. I present an expression system that incorporates elements known to reduce gene expression noise: negative autoregulation and bicistronic transcription. I show by stochastic simulation that while negative autoregulation can produce a more gradual response to induction, bicistronic expression of a repressor and gene of interest can be necessary to reduce noise below the extrinsic limit. I synthesized a plasmid-based system incorporating these principles and studied its properties in Escherichia coli cells, using flow cytometry and fluorescence microscopy to characterize induction dose-response, induction/repression kinetics and gene expression noise. By varying ribosome binding site strengths, expression levels from 55–10,740 molecules/cell were achieved with noise below the extrinsic limit. Individual strains are inducible across a dynamic range greater than 20-fold. Experimental comparison of different regulatory networks confirmed that bicistronic autoregulation reduces noise, and revealed unexpectedly high noise for a conventional expression system with a constitutively expressed transcriptional repressor. I suggest a hybrid, low-noise expression system to increase the dynamic range. PMID:29084263

Generation of stable cell line by using chitosan as gene delivery system.

PubMed

Şalva, Emine; Turan, Suna Özbaş; Ekentok, Ceyda; Akbuğa, Jülide

2016-08-01

Establishing stable cell lines are useful tools to study the function of various genes and silence or induce the expression of a gene of interest. Nonviral gene transfer is generally preferred to generate stable cell lines in the manufacturing of recombinant proteins. In this study, we aimed to establish stable recombinant HEK-293 cell lines by transfection of chitosan complexes preparing with pDNA which contain LacZ and GFP genes. Chitosan which is a cationic polymer was used as gene delivery system. Stable HEK-293 cell lines were established by transfection of cells with complexes which were prepared with chitosan and pVitro-2 plasmid vector that contains neomycin drug resistance gene, beta gal and GFP genes. The transfection efficiency was shown with GFP expression in the cells using fluorescence microscopy. Beta gal protein expression in stable cells was examined by beta-galactosidase assay as enzymatically and X-gal staining method as histochemically. Full complexation was shown in the above of 1/1 ratio in the chitosan/pDNA complexes. The highest beta-galactosidase activity was obtained with transfection of chitosan complexes. Beta gal gene expression was 15.17 ng/ml in the stable cells generated by chitosan complexes. In addition, intensive blue color was observed depending on beta gal protein expression in the stable cell line with X-gal staining. We established a stable HEK-293 cell line that can be used for recombinant protein production or gene expression studies by transfecting the gene of interest.

Mutagenesis Screen Identifies agtpbp1 and eps15L1 as Essential for T lymphocyte Development in Zebrafish.

PubMed

Seiler, Christoph; Gebhart, Nichole; Zhang, Yong; Shinton, Susan A; Li, Yue-sheng; Ross, Nicola L; Liu, Xingjun; Li, Qin; Bilbee, Alison N; Varshney, Gaurav K; LaFave, Matthew C; Burgess, Shawn M; Balciuniene, Jorune; Balciunas, Darius; Hardy, Richard R; Kappes, Dietmar J; Wiest, David L; Rhodes, Jennifer

2015-01-01

Genetic screens are a powerful tool to discover genes that are important in immune cell development and function. The evolutionarily conserved development of lymphoid cells paired with the genetic tractability of zebrafish make this a powerful model system for this purpose. We used a Tol2-based gene-breaking transposon to induce mutations in the zebrafish (Danio rerio, AB strain) genome, which served the dual purpose of fluorescently tagging cells and tissues that express the disrupted gene and provided a means of identifying the disrupted gene. We identified 12 lines in which hematopoietic tissues expressed green fluorescent protein (GFP) during embryonic development, as detected by microscopy. Subsequent analysis of young adult fish, using a novel approach in which single cell suspensions of whole fish were analyzed by flow cytometry, revealed that 8 of these lines also exhibited GFP expression in young adult cells. An additional 15 lines that did not have embryonic GFP+ hematopoietic tissue by microscopy, nevertheless exhibited GFP+ cells in young adults. RT-PCR analysis of purified GFP+ populations for expression of T and B cell-specific markers identified 18 lines in which T and/or B cells were fluorescently tagged at 6 weeks of age. As transposon insertion is expected to cause gene disruption, these lines can be used to assess the requirement for the disrupted genes in immune cell development. Focusing on the lines with embryonic GFP+ hematopoietic tissue, we identified three lines in which homozygous mutants exhibited impaired T cell development at 6 days of age. In two of the lines we identified the disrupted genes, agtpbp1 and eps15L1. Morpholino-mediated knockdown of these genes mimicked the T cell defects in the corresponding mutant embryos, demonstrating the previously unrecognized, essential roles of agtpbp1 and eps15L1 in T cell development.

Constitutive expression of the nifA gene activates associative nitrogen fixation of Enterobacter gergoviae 57-7, an opportunistic endophytic diazotroph.

PubMed

An, Q; Dong, Y; Wang, W; Li, Y; Li, J

2007-09-01

This study was undertaken to investigate whether a nitrogen-fixing bacterium Enterobacter gergoviae 57-7, which was isolated from surface-sterilized maize (Zea mays L.) roots, can colonize in roots and whether constitutive expression of the nifA gene encoding the transcriptional activator of nitrogenase genes can activate nif gene expression in planta. Maize seedlings grown in an agar medium were inoculated with Ent. gergoviae strains containing the green fluorescent protein reporter gene. Root colonization and expression of the dinitrogenase reductase gene (nifH) by Ent. gergoviae were observed by confocal laser scanning microscopy. gfp-tagged Ent. gergoviae was observed to colonize predominantly in cortical aerenchyma of primary and lateral roots and in stellar parenchyma cells and xylem vessels of primary roots. In planta nifH :: gfp expression was not detected but after a constitutively expressed nifA gene was introduced into Ent. gergoviae. Enterobacter gergoviae 57-7 is an opportunistic endophyte because it can live in soil and colonize in maize roots in the gnotobiotic agar culture. In agreement with previous (15)N-dilution evidence that Ent. gergoviae 57-7 did not fix N(2) in association with maize in pots whereas a derivative E7 containing a constitutively expressed nifA gene promoted plant growth partly through associative nitrogen fixation, constitutive expression of the nifA gene can activate bacterial nif gene expression in planta. This study and our previous studies suggest that manipulation of the promoter of the nifA gene in a nitrogen-fixing bacterium having a high colonization competence is a practical and promising approach to achieve a stable associative nitrogen fixation for cereals.

A software solution for recording circadian oscillator features in time-lapse live cell microscopy.

PubMed

Sage, Daniel; Unser, Michael; Salmon, Patrick; Dibner, Charna

2010-07-06

Fluorescent and bioluminescent time-lapse microscopy approaches have been successfully used to investigate molecular mechanisms underlying the mammalian circadian oscillator at the single cell level. However, most of the available software and common methods based on intensity-threshold segmentation and frame-to-frame tracking are not applicable in these experiments. This is due to cell movement and dramatic changes in the fluorescent/bioluminescent reporter protein during the circadian cycle, with the lowest expression level very close to the background intensity. At present, the standard approach to analyze data sets obtained from time lapse microscopy is either manual tracking or application of generic image-processing software/dedicated tracking software. To our knowledge, these existing software solutions for manual and automatic tracking have strong limitations in tracking individual cells if their plane shifts. In an attempt to improve existing methodology of time-lapse tracking of a large number of moving cells, we have developed a semi-automatic software package. It extracts the trajectory of the cells by tracking theirs displacements, makes the delineation of cell nucleus or whole cell, and finally yields measurements of various features, like reporter protein expression level or cell displacement. As an example, we present here single cell circadian pattern and motility analysis of NIH3T3 mouse fibroblasts expressing a fluorescent circadian reporter protein. Using Circadian Gene Express plugin, we performed fast and nonbiased analysis of large fluorescent time lapse microscopy datasets. Our software solution, Circadian Gene Express (CGE), is easy to use and allows precise and semi-automatic tracking of moving cells over longer period of time. In spite of significant circadian variations in protein expression with extremely low expression levels at the valley phase, CGE allows accurate and efficient recording of large number of cell parameters, including level of reporter protein expression, velocity, direction of movement, and others. CGE proves to be useful for the analysis of widefield fluorescent microscopy datasets, as well as for bioluminescence imaging. Moreover, it might be easily adaptable for confocal image analysis by manually choosing one of the focal planes of each z-stack of the various time points of a time series. CGE is a Java plugin for ImageJ; it is freely available at: http://bigwww.epfl.ch/sage/soft/circadian/.

Effects of Ghrelin miRNA on Inflammation and Calcium Pathway in Pancreatic Acinar Cells of Acute Pancreatitis.

PubMed

Tang, Xiping; Tang, Guodu; Liang, Zhihai; Qin, Mengbin; Fang, Chunyun; Zhang, Luyi

The study investigated the effects of endogenous targeted inhibition of ghrelin gene on inflammation and calcium pathway in an in vitro pancreatic acinar cell model of acute pancreatitis. Lentiviral expression vector against ghrelin gene was constructed and transfected into AR42J cells. The mRNA and protein expression of each gene were detected by reverse transcription polymerase chain reaction, Western blotting, or enzyme-linked immunosorbent assay. The concentration of intracellular calcium ([Ca]i) was determined by calcium fluorescence mark probe combined with laser scanning confocal microscopy. Compared with the control group, cerulein could upregulate mRNA and protein expression of inflammatory factors, calcium pathway, ghrelin, and [Ca]i. mRNA and protein expression of inflammatory factors increased significantly in cells transfected with ghrelin miRNA compared with the other groups. Intracellular calcium and expression of some calcium pathway proteins decreased significantly in cells transfected with ghrelin miRNA compared with the other groups. Targeted inhibition of ghrelin gene in pancreatic acinar cells of acute pancreatitis can upregulate the expression of the intracellular inflammatory factors and alleviate the intracellular calcium overload.

Crx broadly modulates the pineal transcriptome

PubMed Central

Rovsing, Louise; Clokie, Samuel; Bustos, Diego M.; Rohde, Kristian; Coon, Steven L.; Litman, Thomas; Rath, Martin F.; Møller, Morten; Klein, David C.

2011-01-01

Cone-rod homeobox (Crx) encodes Crx, a transcription factor expressed selectively in retinal photoreceptors and pinealocytes, the major cell type of the pineal gland. Here, the influence of Crx on the mammalian pineal gland was studied by light and electron microscopy and by use of microarray and qRTPCR technology, thereby extending previous studies on selected genes (Furukawa et al. 1999). Deletion of Crx was not found to alter pineal morphology, but was found to broadly modulate the mouse pineal transcriptome, characterized by a >2-fold downregulation of 543 genes and a >2-fold upregulation of 745 genes (p < 0.05). Of these, one of the most highly upregulated (18-fold) is Hoxc4, a member of the Hox gene family, members of which are known to control gene expression cascades. During a 24-hour period, a set of 51 genes exhibited differential day/night expression in pineal glands of wild-type animals; only eight of these were also day/night expressed in the Crx−/− pineal gland. However, in the Crx−/− pineal gland 41 genes exhibit differential night/day expression that is not seen in wild-type animals. These findings indicate that Crx broadly modulates the pineal transcriptome and also influences differential night/day gene expression in this tissue. Some effects of Crx deletion on the pineal transcriptome might be mediated by Hoxc4 upregulation. PMID:21797868

Gene expression and morphogenesis during the deposition of Drosophila wing cuticle

PubMed Central

Adler, Paul N.

2017-01-01

ABSTRACT The exoskeleton of insects and other arthropods is a very versatile material that is characterized by a complex multilayer structure. In Sobala and Adler (2016) we analyzed the process of wing cuticle deposition by RNAseq and electron microscopy. In this extra view we discuss the unique aspects of the envelope the first and most outermost layer and the gene expression program seen at the end of cuticle deposition. We discussed the role of undulae in the deposition of cuticle and how the hydrophobicity of wing cuticle arises. PMID:28631994

Gene expression and morphogenesis during the deposition of Drosophila wing cuticle.

PubMed

Adler, Paul N

2017-07-03

The exoskeleton of insects and other arthropods is a very versatile material that is characterized by a complex multilayer structure. In Sobala and Adler (2016) we analyzed the process of wing cuticle deposition by RNAseq and electron microscopy. In this extra view we discuss the unique aspects of the envelope the first and most outermost layer and the gene expression program seen at the end of cuticle deposition. We discussed the role of undulae in the deposition of cuticle and how the hydrophobicity of wing cuticle arises.

MATER protein expression and intracellular localization throughout folliculogenesis and preimplantation embryo development in the bovine

PubMed Central

Pennetier, Sophie; Perreau, Christine; Uzbekova, Svetlana; Thélie, Aurore; Delaleu, Bernadette; Mermillod, Pascal; Dalbiès-Tran, Rozenn

2006-01-01

Background Mater (Maternal Antigen that Embryos Require), also known as Nalp5 (NACHT, leucine rich repeat and PYD containing 5), is an oocyte-specific maternal effect gene required for early embryonic development beyond the two-cell stage in mouse. We previously characterized the bovine orthologue MATER as an oocyte marker gene in cattle, and this gene was recently assigned to a QTL region for reproductive traits. Results Here we have analyzed gene expression during folliculogenesis and preimplantation embryo development. In situ hybridization and immunohistochemistry on bovine ovarian section revealed that both the transcript and protein are restricted to the oocyte from primary follicles onwards, and accumulate in the oocyte cytoplasm during follicle growth. In immature oocytes, cytoplasmic, and more precisely cytosolic localization of MATER was confirmed by immunohistochemistry coupled with confocal microscopy and immunogold electron microscopy. By real-time PCR, MATER messenger RNA was observed to decrease strongly during maturation, and progressively during the embryo cleavage stages; it was hardly detected in morulae and blastocysts. The protein persisted after fertilization up until the blastocyst stage, and was mostly degraded after hatching. A similar predominantly cytoplasmic localization was observed in blastomeres from embryos up to 8-cells, with an apparent concentration near the nuclear membrane. Conclusion Altogether, these expression patterns are consistent with bovine MATER protein being an oocyte specific maternal effect factor as in mouse. PMID:16753072

Visualisation of the mechanosensitive channel of large conductance in bacteria using confocal microscopy.

PubMed

Norman, Christel; Liu, Zhen-Wei; Rigby, Paul; Raso, Albert; Petrov, Yevgeniy; Martinac, Boris

2005-07-01

The mechanosensitive channel of large conductance (MscL) plays an important role in the survival of bacterial cells to hypo-osmotic shock. This channel has been extensively studied and its sequence, structure and electrophysiological characteristics are well known. Here we present a method to visualise MscL in living bacteria using confocal microscopy. By creating a gene fusion between mscl and the gene encoding the green fluorescent protein (GFP) we were able to express the fusion protein MscL-GFP in bacteria. We show that MscL-GFP is present in the cytoplasmic membrane and forms functional channels. These channels have the same characteristics as wild-type MscL, except that they require more pressure to open. This method could prove an interesting, non-invasive, tool to study the localisation and the regulation of expression of MscL in bacteria.

Fear conditioning leads to alteration in specific genes expression in cortical and thalamic neurons that project to the lateral amygdala.

PubMed

Katz, Ira K; Lamprecht, Raphael

2015-02-01

RNA transcription is needed for memory formation. However, the ability to identify genes whose expression is altered by learning is greatly impaired because of methodological difficulties in profiling gene expression in specific neurons involved in memory formation. Here, we report a novel approach to monitor the expression of genes after learning in neurons in specific brain pathways needed for memory formation. In this study, we aimed to monitor gene expression after fear learning. We retrogradely labeled discrete thalamic neurons that project to the lateral amygdala (LA) of rats. The labeled neurons were dissected, using laser microdissection microscopy, after fear conditioning learning or unpaired training. The RNAs from the dissected neurons were subjected to microarray analysis. The levels of selected RNAs detected by the microarray analysis to be altered by fear conditioning were also assessed by nanostring analysis. We observed that the expression of genes involved in the regulation of translation, maturation and degradation of proteins was increased 6 h after fear conditioning compared to unpaired or naïve trained rats. These genes were not expressed 24 h after training or in cortical neurons that project to the LA. The expression of genes involved in transcription regulation and neuronal development was altered after fear conditioning learning in the cortical-LA pathway. The present study provides key information on the identity of genes expressed in discrete thalamic and cortical neurons that project to the LA after fear conditioning. Such an approach could also serve to identify gene products as targets for the development of a new generation of therapeutic agents that could be aimed to functionally identified brain circuits to treat memory-related disorders. © 2014 International Society for Neurochemistry.

Quantitative gene expression analysis in Caenorhabditis elegans using single molecule RNA FISH.

PubMed

Bolková, Jitka; Lanctôt, Christian

2016-04-01

Advances in fluorescent probe design and synthesis have allowed the uniform in situ labeling of individual RNA molecules. In a technique referred to as single molecule RNA FISH (smRNA FISH), the labeled RNA molecules can be imaged as diffraction-limited spots and counted using image analysis algorithms. Single RNA counting has provided valuable insights into the process of gene regulation. This microscopy-based method has often revealed a high cell-to-cell variability in expression levels, which has in turn led to a growing interest in investigating the biological significance of gene expression noise. Here we describe the application of the smRNA FISH technique to samples of Caenorhabditis elegans, a well-characterized model organism. Copyright © 2015 Elsevier Inc. All rights reserved.

5D imaging via light sheet microscopy reveals cell dynamics during the eye-antenna disc primordium formation in Drosophila

NASA Astrophysics Data System (ADS)

Huang, Yu Shan; Ku, Hui Yu; Tsai, Yun Chi; Chang, Chin Hao; Pao, Sih Hua; Sun, Y. Henry; Chiou, Arthur

2017-03-01

5D images of engrailed (en) and eye gone (eyg) gene expressions during the course of the eye-antenna disc primordium (EADP) formation of Drosophila embryos from embryonic stages 13 through 16 were recorded via light sheet microscopy and analyzed to reveal the cell dynamics involved in the development of the EADP. Detailed analysis of the time-lapsed images revealed the process of EADP formation and its invagination trajectory, which involved an inversion of the EADP anterior-posterior axis relative to the body. Furthermore, analysis of the en-expression pattern in the EADP provided strong evidence that the EADP is derived from one of the en-expressing head segments.

[Establishment of RAW264.7 cell strain stably expressing RFP-GFP-LC3].

PubMed

Wang, Wan; Zhang, Qing; Zhao, Runpeng; Xu, Xuewei; Xing, Yingru; Zhang, Rongbo; Wu, Jing; Hu, Dong

2015-09-01

To establish murine macrophage RAW264.7 cell strain with stable expression of red fluorescent protein-green fluorescent protein-microtubule associated protein light chain 3 (RFP-GFP-LC3). A lentiviral vector containing RFP-GFP-LC3 gene was constructed and then packaged in HEK293T cells with the packaging plasmids. The viral supernatant was collected to infect RAW264.7 cells. The RAW264.7 cell strain with stable expression of RFP-GFP-LC3 was screened with puromycin and analyzed with flow cytometry and fluorescent microscopy for infection efficiency. The number of RFP-GFP-LC3 puncta was observed using florescence microscopy following starvation treatment. The recombinant lentivirus pLV-CMV-RFP-GFP-LC3 was successfully constructed. The RAW264.7 cells with stable expression of RFP-GFP-LC3 were obtained by viral infection and puromycin screening. Fluorescent microscopy and flow cytometry demonstrated the expression rates of RFP and GFP reached to 100%. The number of autophagic puncta significantly increased after starvation treatment. The RAW264.7 cell strain with stable expression of RFP-GFP-LC3 has been successfully constructed, which provides a reliable cellular platform for autophagy research.

Identification of Genes Expressed in Premalignant Breast Disease by Microscopy-Directed Cloning

NASA Astrophysics Data System (ADS)

Jensen, Roy A.; Page, David L.; Holt, Jeffrey T.

1994-09-01

Histopathologic study of human breast biopsy samples has identified specific lesions which are associated with a high risk of development of invasive breast cancer. Presumably, these lesions (collectively termed premalignant breast disease) represent the earliest recognizable morphologic expression of fundamental molecular events that lead to the development of invasive breast cancer. To study molecular events underlying premalignant breast disease, we have developed a method for isolating RNA from histologically identified lesions from frozen human breast tissue. This method specifically obtains mRNA from breast epithelial cells and has identified three genes which are differentially expressed in premalignant breast epithelial lesions. One gene identified by this method is overexpressed in four of five noncomedo ductal carcinoma in situ lesions and appears to be the human homologue of the gene encoding the M2 subunit of ribonucleotide reductase, an enzyme involved in DNA synthesis.

In vivo imaging of induction of heat-shock protein-70 gene expression with fluorescence reflectance imaging and intravital confocal microscopy following brain ischaemia in reporter mice.

PubMed

de la Rosa, Xavier; Santalucía, Tomàs; Fortin, Pierre-Yves; Purroy, Jesús; Calvo, Maria; Salas-Perdomo, Angélica; Justicia, Carles; Couillaud, Franck; Planas, Anna M

2013-02-01

Stroke induces strong expression of the 72-kDa heat-shock protein (HSP-70) in the ischaemic brain, and neuronal expression of HSP-70 is associated with the ischaemic penumbra. The aim of this study was to image induction of Hsp-70 gene expression in vivo after brain ischaemia using reporter mice. A genomic DNA sequence of the Hspa1b promoter was used to generate an Hsp70-mPlum far-red fluorescence reporter vector. The construct was tested in cellular systems (NIH3T3 mouse fibroblast cell line) by transient transfection and examining mPlum and Hsp-70 induction under a challenge. After construct validation, mPlum transgenic mice were generated. Focal brain ischaemia was induced by transient intraluminal occlusion of the middle cerebral artery and the mice were imaged in vivo with fluorescence reflectance imaging (FRI) with an intact skull, and with confocal microscopy after opening a cranial window. Cells transfected with the Hsp70-mPlum construct showed mPlum fluorescence after stimulation. One day after induction of ischaemia, reporter mice showed a FRI signal located in the HSP-70-positive zone within the ipsilateral hemisphere, as validated by immunohistochemistry. Live confocal microscopy allowed brain tissue to be visualized at the cellular level. mPlum fluorescence was observed in vivo in the ipsilateral cortex 1 day after induction of ischaemia in neurons, where it is compatible with penumbra and neuronal viability, and in blood vessels in the core of the infarction. This study showed in vivo induction of Hsp-70 gene expression in ischaemic brain using reporter mice. The fluorescence signal showed in vivo the induction of Hsp-70 in penumbra neurons and in the vasculature within the ischaemic core.

BcMF8, a putative arabinogalactan protein-encoding gene, contributes to pollen wall development, aperture formation and pollen tube growth in Brassica campestris

PubMed Central

Lin, Sue; Dong, Heng; Zhang, Fang; Qiu, Lin; Wang, Fangzhan; Cao, Jiashu; Huang, Li

2014-01-01

Background and Aims The arabinogalactan protein (AGP) gene family is involved in plant reproduction. However, little is known about the function of individual AGP genes in pollen development and pollen tube growth. In this study, Brassica campestris male fertility 8 (BcMF8), a putative AGP-encoding gene previously found to be pollen specific in Chinese cabbage (B. campestris ssp. chinensis), was investigated. Methods Real-time reverse transcription–PCR and in situ hybridization were used to analyse the expression pattern of BcMF8 in pistils. Prokaryotic expression and western blots were used to ensure that BcMF8 could encode a protein. Antisense RNA technology was applied to silence gene expression, and morphological and cytological approaches (e.g. scanning electron microscopy and transmission electron microscopy) were used to reveal abnormal phenotypes caused by gene silencing. Key Results The BcMF8 gene encoded a putative AGP protein that was located in the cell wall, and was expressed in pollen grains and pollen tubes. The functional interruption of BcMF8 by antisense RNA technology resulted in slipper-shaped and bilaterally sunken pollen with abnormal intine development and aperture formation. The inhibition of BcMF8 led to a decrease in the percentage of in vitro pollen germination. In pollen that did germinate, the pollen tubes were unstable, abnormally shaped and burst more frequently relative to controls, which corresponded to an in vivo arrest of pollen germination at the stigma surface and retarded pollen tube growth in the stylar transmitting tissues. Conclusions The phenotypic defects of antisense BcMF8 RNA lines (bcmf8) suggest a crucial function of BcMF8 in modulating the physical nature of the pollen wall and in helping in maintaining the integrity of the pollen tube wall matrix. PMID:24489019

Neuroprotective effects of diazoxide and its antagonism by glibenclamide in pyramidal neurons of rat hippocampus subjected to ischemia-reperfusion-induced injury.

PubMed

Zarch, Anoushiravan Vakili; Toroudi, Hamidreza Pazoki; Soleimani, Mansooreh; Bakhtiarian, Azam; Katebi, Majid; Djahanguiri, Bijan

2009-01-01

Mitochondrial ATP-sensitive potassium channel opener, diazoxide, is shown to have protective effect on the heart and brain following ischemia-reperfusion-induced injury (IR/II). However, the detailed effect of diazoxide and its antagonist on neuronal death, mitochondrial changes, and apoptosis in cerebral IR/II has not fully studied. IR/II was induced in rats by the 4-vessel occlusion model. Neuronal cell death and mitochondrial changes in CA1-CA4 pyramidal cells of the hippocampus were studied by light and electron microscopy, respectively. Apoptosis was assessed by measuring the amount of protein expressed by Bax and Bcl-2 genes. In light microscopy studies, the number of total and normal cells were increased only following 18 mg/kg of diazoxide. Lower doses (2 and 6 mg/kg) failed to change the cell numbers. All three doses of glibenclamide (1, 5, and 25 mg/kg) decreased the number of total and normal cell populations. In electron microscopy studies, different doses of diazoxide and glibenclamide prevented and aggravated the IR-induced morphological changes, respectively. Western blot analysis showed that diazoxide and glibenclamide inhibited and enhanced Bax protein expression respectively. Regarding Bcl-2 expression, only diazoxide showed a significant enhancement of gene expression. In conclusion, the results show that diazoxide can exhibit neuroprotective effects against IR/II in hippocampal regions, possibly through the opening of mitochondrial ATP-sensitive K(+) channels.

Principles of Systems Biology, No. 29.

PubMed

2018-05-23

This month: in silico labeling of microscopy images (Christiansen/Finkbeiner), single-cell lineage trees and data integration (Rajewsky, Satija), gene expression (Weinberger/Simpson, Tavazoie, Ameres/Zuber), and signalling networks (Mercer/Wollscheid, Fussenegger). Copyright © 2018. Published by Elsevier Inc.

Application of unstable Gfp variants to the kinetic study of Legionella pneumophila icm gene expression during infection.

PubMed

Barysheva, Oksana V; Fujii, Jun; Takaesu, Giichi; Yoshida, Shin-ichi

2008-04-01

An unstable type of green fluorescent protein (Gfp) tagged with a C-terminal extension, which is a target for tail-specific protease, was used as a reporter gene in Legionella pneumophila. To analyse Gfp expression in legionellae, transcriptional fusions of unstable gfp with the Legionella-specific icm (intracellular multiplication) promoters (P(icmS), P(icmT) and P(icmQ)) were constructed. Infection studies using J774.1 macrophages as the host, and L. pneumophila strains carrying P(icmS)-gfp, P(icmT)-gfp and P(icmQ)-gfp fusions, indicated that the icmS, icmT and icmQ genes could be expressed intracellularly. Expression of icmS, icmT and icmQ genes in infected cells was examined by flow cytometry. Furthermore, fluorescent intracellular legionellae were detected directly by confocal microscopy. Real-time quantitative RT-PCR revealed the differences in the gene expression of icmS, and that of icmT and icmQ, during infection. Expression of icmS was high in the late stage of infection, while that of icmT and icmQ was high in the early phase only. We show that unstable gfp is a useful reporter gene whose expression in legionellae can be followed in real-time, and that it allows analysis of promoter activities in legionellae and monitoring of the infection process.

Analysis of the neuronal marker protein gene product 9.5 in internal limiting membranes after indocyanine-green assisted peeling.

PubMed

Peters, Swaantje; Tatar, Olcay; Spitzer, Martin S; Szurman, Peter; Aisenbrey, Sabine; Lüke, Matthias; Adam, Annemarie; Yoeruek, Efdal; Grisanti, Salvatore

2009-02-01

Indocyanine green-assisted internal limiting membrane (ILM) peeling was suspected to disrupt the innermost layer of the neural retina. We examined whether surgically excised specimens contain remnants of neuronal tissue. Ten patients with macular hole underwent pars plana vitrectomy and indocyanine green-assisted ILM peeling. A total of 0.1 mL of a 0.5% indocyanine green solution was applied for 15 seconds. The ILM specimens were prepared for immunohistochemistry, using a polyclonal antibody against protein gene product 9.5. Protein gene product 9.5 is a pan-neuronal marker labeling human neuronal cells. Appropriate controls to show selectivity of the antibody were performed on neuronal tissue of donor eyes. One ILM was prepared for electron microscopy. A selective expression of protein gene product 9.5 was found in neuronal fibers of the retina and optic nerve of donor eyes. Only 1 of the 10 surgical ILM specimens showed a minimal focal positivity for protein gene product 9.5. No neuronal tissue was detected on the ILM by electron microscopy. Focal expression of protein gene product 9.5 in only 1 of 10 surgical ILM specimens argues against a general indocyanine green-related disruption of the innermost retinal layers. However, higher concentrations of the dye, longer incubation times or different solvents than used in this study may lead to different results.

Combining laser microdissection and RNA-seq to chart the transcriptional landscape of fungal development

PubMed Central

2012-01-01

Background During sexual development, filamentous ascomycetes form complex, three-dimensional fruiting bodies for the protection and dispersal of sexual spores. Fruiting bodies contain a number of cell types not found in vegetative mycelium, and these morphological differences are thought to be mediated by changes in gene expression. However, little is known about the spatial distribution of gene expression in fungal development. Here, we used laser microdissection (LM) and RNA-seq to determine gene expression patterns in young fruiting bodies (protoperithecia) and non-reproductive mycelia of the ascomycete Sordaria macrospora. Results Quantitative analysis showed major differences in the gene expression patterns between protoperithecia and total mycelium. Among the genes strongly up-regulated in protoperithecia were the pheromone precursor genes ppg1 and ppg2. The up-regulation was confirmed by fluorescence microscopy of egfp expression under the control of ppg1 regulatory sequences. RNA-seq analysis of protoperithecia from the sterile mutant pro1 showed that many genes that are differentially regulated in these structures are under the genetic control of transcription factor PRO1. Conclusions We have generated transcriptional profiles of young fungal sexual structures using a combination of LM and RNA-seq. This allowed a high spatial resolution and sensitivity, and yielded a detailed picture of gene expression during development. Our data revealed significant differences in gene expression between protoperithecia and non-reproductive mycelia, and showed that the transcription factor PRO1 is involved in the regulation of many genes expressed specifically in sexual structures. The LM/RNA-seq approach will also be relevant to other eukaryotic systems in which multicellular development is investigated. PMID:23016559

The biosynthetic gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor contains its co-expressed vacuolar MATE transporter

PubMed Central

Darbani, Behrooz; Motawia, Mohammed Saddik; Olsen, Carl Erik; Nour-Eldin, Hussam H.; Møller, Birger Lindberg; Rook, Fred

2016-01-01

Genomic gene clusters for the biosynthesis of chemical defence compounds are increasingly identified in plant genomes. We previously reported the independent evolution of biosynthetic gene clusters for cyanogenic glucoside biosynthesis in three plant lineages. Here we report that the gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor additionally contains a gene, SbMATE2, encoding a transporter of the multidrug and toxic compound extrusion (MATE) family, which is co-expressed with the biosynthetic genes. The predicted localisation of SbMATE2 to the vacuolar membrane was demonstrated experimentally by transient expression of a SbMATE2-YFP fusion protein and confocal microscopy. Transport studies in Xenopus laevis oocytes demonstrate that SbMATE2 is able to transport dhurrin. In addition, SbMATE2 was able to transport non-endogenous cyanogenic glucosides, but not the anthocyanin cyanidin 3-O-glucoside or the glucosinolate indol-3-yl-methyl glucosinolate. The genomic co-localisation of a transporter gene with the biosynthetic genes producing the transported compound is discussed in relation to the role self-toxicity of chemical defence compounds may play in the formation of gene clusters. PMID:27841372

Medium-throughput processing of whole mount in situ hybridisation experiments into gene expression domains.

PubMed

Crombach, Anton; Cicin-Sain, Damjan; Wotton, Karl R; Jaeger, Johannes

2012-01-01

Understanding the function and evolution of developmental regulatory networks requires the characterisation and quantification of spatio-temporal gene expression patterns across a range of systems and species. However, most high-throughput methods to measure the dynamics of gene expression do not preserve the detailed spatial information needed in this context. For this reason, quantification methods based on image bioinformatics have become increasingly important over the past few years. Most available approaches in this field either focus on the detailed and accurate quantification of a small set of gene expression patterns, or attempt high-throughput analysis of spatial expression through binary pattern extraction and large-scale analysis of the resulting datasets. Here we present a robust, "medium-throughput" pipeline to process in situ hybridisation patterns from embryos of different species of flies. It bridges the gap between high-resolution, and high-throughput image processing methods, enabling us to quantify graded expression patterns along the antero-posterior axis of the embryo in an efficient and straightforward manner. Our method is based on a robust enzymatic (colorimetric) in situ hybridisation protocol and rapid data acquisition through wide-field microscopy. Data processing consists of image segmentation, profile extraction, and determination of expression domain boundary positions using a spline approximation. It results in sets of measured boundaries sorted by gene and developmental time point, which are analysed in terms of expression variability or spatio-temporal dynamics. Our method yields integrated time series of spatial gene expression, which can be used to reverse-engineer developmental gene regulatory networks across species. It is easily adaptable to other processes and species, enabling the in silico reconstitution of gene regulatory networks in a wide range of developmental contexts.

Are specific gene expressions of extracellular matrix and nucleus pulposus affected by primary cell cultures prepared from intact or degenerative intervertebral disc tissues?

PubMed

Karaarslan, Numan; Yilmaz, Ibrahim; Ozbek, Hanefi; Sirin Yasar, Duygu; Kaplan, Necati; Akyuva, Yener; Gonultas, Aylin; Ates, Ozkan

2018-01-22

In this scientific research project, the researchers aimed to determine the gene expression patterns of nucleus pulposus (NP) in cell cultures obtained from degenerated or intact tissues. Whereas 12 of the cases were diagnosed with lumbar disc hernia and had undergone lumbar microdiscectomy, 12 cases had undergone traumatic intervertebral discectomy and corpectomy, along with discectomy after spinal trauma. NP-specific markers and gene expressions of the reagents of the extracellular matrix in the experimental setup were tested at the 0th, 24th, and 48th hours by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Visual evaluations were simultaneously made in all samples using invert and fluorescence microscopy. Vitality and proliferation analyses were evaluated by UV spectrophotometer. As a method of statistical evaluation, Spearman was used for categorical variants, and the Pearson correlation was used for variants with numerical and plain distribution. No association was found either between the tissue type and times (r=0.000; p=1.000) or between the region that the tissue was obtained from and hypoxia transcription factor-1 alpha (HIF-1α) gene expression (r=0.098; p=0.245). There was no correlation between cell proliferation and chondroadherin (CHAD) expression or between type II collagen (COL2A1) and CHAD gene expressions. It was found that CHAD and HIF-1α gene expressions and HIF-1α and COL2A1 gene expressions affected cell proliferation. Cell culture setups are of paramount importance because they may influence the pattern of changes in the gene expressions of the cells used in these setups.

Oil Bodies and Oleosins in Physcomitrella Possess Characteristics Representative of Early Trends in Evolution1[W][OA

PubMed Central

Huang, Chien-Yu; Chung, Chun-I; Lin, Yao-Cheng; Hsing, Yue-Ie Caroline; Huang, Anthony H.C.

2009-01-01

Searches of sequenced genomes of diverse organisms revealed that the moss Physcomitrella patens is the most primitive organism possessing oleosin genes. Microscopy examination of Physcomitrella revealed that oil bodies (OBs) were abundant in the photosynthetic vegetative gametophyte and the reproductive spore. Chromatography illustrated the neutral lipids in OBs isolated from the gametophyte to be largely steryl esters and triacylglycerols, and SDS-PAGE showed the major proteins to be oleosins. Reverse transcription-PCR revealed the expression of all three oleosin genes to be tissue specific. This tissue specificity was greatly altered via alternative splicing, a control mechanism of oleosin gene expression unknown in higher plants. During the production of sex organs at the tips of gametophyte branches, the number of OBs in the top gametophyte tissue decreased concomitant with increases in the number of peroxisomes and level of transcripts encoding the glyoxylate cycle enzymes; thus, the OBs are food reserves for gluconeogenesis. In spores during germination, peroxisomes adjacent to OBs, along with transcripts encoding the glyoxylate cycle enzymes, appeared; thus, the spore OBs are food reserves for gluconeogenesis and equivalent to seed OBs. The one-cell-layer gametophyte could be observed easily with confocal microscopy for the subcellular OBs and other structures. Transient expression of various gene constructs transformed into gametophyte cells revealed that all OBs were linked to the endoplasmic reticulum (ER), that oleosins were synthesized in extended regions of the ER, and that two different oleosins were colocated in all OBs. PMID:19420327

Early development of Moniliophthora perniciosa basidiomata and developmentally regulated genes

PubMed Central

2009-01-01

Background The hemibiotrophic fungus Moniliophthora perniciosa is the causal agent of Witches' broom, a disease of Theobroma cacao. The pathogen life cycle ends with the production of basidiocarps in dead tissues of the infected host. This structure generates millions of basidiospores that reinfect young tissues of the same or other plants. A deeper understanding of the mechanisms underlying the sexual phase of this fungus may help develop chemical, biological or genetic strategies to control the disease. Results Mycelium was morphologically analyzed prior to emergence of basidiomata by stereomicroscopy, light microscopy and scanning electron microscopy. The morphological changes in the mycelium before fructification show a pattern similar to other members of the order Agaricales. Changes and appearance of hyphae forming a surface layer by fusion were correlated with primordia emergence. The stages of hyphal nodules, aggregation, initial primordium and differentiated primordium were detected. The morphological analysis also allowed conclusions on morphogenetic aspects. To analyze the genes involved in basidiomata development, the expression of some selected EST genes from a non-normalized cDNA library, representative of the fruiting stage of M. perniciosa, was evaluated. A macroarray analysis was performed with 192 selected clones and hybridized with two distinct RNA pools extracted from mycelium in different phases of basidiomata formation. This analysis showed two groups of up and down-regulated genes in primordial phases of mycelia. Hydrophobin coding, glucose transporter, Rho-GEF, Rheb, extensin precursor and cytochrome p450 monooxygenase genes were grouped among the up-regulated. In the down-regulated group relevant genes clustered coding calmodulin, lanosterol 14 alpha demethylase and PIM1. In addition, 12 genes with more detailed expression profiles were analyzed by RT-qPCR. One aegerolysin gene had a peak of expression in mycelium with primordia and a second in basidiomata, confirming their distinctiveness. The number of transcripts of the gene for plerototolysin B increased in reddish-pink mycelium and indicated an activation of the initial basidiomata production even at this culturing stage. Expression of the glucose transporter gene increased in mycelium after the stress, coinciding with a decrease of adenylate cyclase gene transcription. This indicated that nutrient uptake can be an important signal to trigger fruiting in this fungus. Conclusion The identification of genes with increased expression in this phase of the life cycle of M. perniciosa opens up new possibilities of controlling fungus spread as well as of genetic studies of biological processes that lead to basidiomycete fruiting. This is the first comparative morphologic study of the early development both in vivo and in vitro of M. perniciosa basidiomata and the first description of genes expressed at this stage of the fungal life cycle. PMID:19653910

Sensitivity of Aspergillus nidulans to the Cellulose Synthase Inhibitor Dichlobenil: Insights from Wall-Related Genes’ Expression and Ultrastructural Hyphal Morphologies

PubMed Central

Obersriebnig, Michael; Salerno, Marco; Pum, Dietmar; Strauss, Joseph

2013-01-01

The fungal cell wall constitutes an important target for the development of antifungal drugs, because of its central role in morphogenesis, development and determination of fungal-specific molecular features. Fungal walls are characterized by a network of interconnected glycoproteins and polysaccharides, namely α-, β-glucans and chitin. Cell walls promptly and dynamically respond to environmental stimuli by a signaling mechanism, which triggers, among other responses, modulations in wall biosynthetic genes’ expression. Despite the absence of cellulose in the wall of the model filamentous fungus Aspergillus nidulans, we found in this study that fungal growth, spore germination and morphology are affected by the addition of the cellulose synthase inhibitor dichlobenil. Expression analysis of selected genes putatively involved in cell wall biosynthesis, carried out at different time points of drug exposure (i.e. 0, 1, 3, 6 and 24 h), revealed increased expression for the putative mixed linkage β-1,3;1,4 glucan synthase celA together with the β-1,3-glucan synthase fksA and the Rho-related GTPase rhoA. We also compared these data with the response to Congo Red, a known plant/fungal drug affecting both chitin and cellulose biosynthesis. The two drugs exerted different effects at the cell wall level, as shown by gene expression analysis and the ultrastructural features observed through atomic force microscopy and scanning electron microscopy. Although the concentration of dichlobenil required to affect growth of A. nidulans is approximately 10-fold higher than that required to inhibit plant cellulose biosynthesis, our work for the first time demonstrates that a cellulose biosynthesis inhibitor affects fungal growth, changes fungal morphology and expression of genes connected to fungal cell wall biosynthesis. PMID:24312197

Up-regulation of mRNA ventricular PRNP prion protein gene expression in air pollution highly exposed young urbanites: endoplasmic reticulum stress, glucose regulated protein 78, and nanosized particles.

PubMed

Villarreal-Calderon, Rodolfo; Franco-Lira, Maricela; González-Maciel, Angélica; Reynoso-Robles, Rafael; Harritt, Lou; Pérez-Guillé, Beatriz; Ferreira-Azevedo, Lara; Drecktrah, Dan; Zhu, Hongtu; Sun, Qiang; Torres-Jardón, Ricardo; Aragón-Flores, Mariana; Calderón-Garcidueñas, Ana; Diaz, Philippe; Calderón-Garcidueñas, Lilian

2013-11-28

Mexico City Metropolitan Area children and young adults exposed to high concentrations of air pollutants including fine and ultrafine particulate matter (PM) vs. clean air controls, exhibit myocardial inflammation and inflammasome activation with a differential right and left ventricular expression of key inflammatory genes and inflammasomes. We investigated the mRNA expression levels of the prion protein gene PRNP, which plays an important role in the protection against oxidative stress and metal toxicity, and the glucose regulated protein 78, a key protein in endoplasmic reticulum (ER) stress signaling, in ventricular autopsy samples from 30 children and young adults age 19.97 ± 6.8 years with a lifetime of low (n:4) vs. high (n:26) air pollution exposures. Light microscopy and transmission electron microscopy studies were carried out in human ventricles, and electron microscopy studies were also done in 5 young, highly exposed Mexico City dogs. There was significant left ventricular PRNP and bi-ventricular GRP78 mRNA up-regulation in Mexico City young urbanites vs. controls. PRNP up-regulation in the left ventricle was significantly different from the right, p < 0.0001, and there was a strong left ventricular PRNP and GRP78 correlation (p = 0.0005). Marked abnormalities in capillary endothelial cells, numerous nanosized particles in myocardial ER and in abnormal mitochondria characterized the highly exposed ventricles. Early and sustained cardiac ER stress could result in detrimental irreversible consequences in urban children, and while highly complex systems maintain myocardial homeostasis, failure to compensate for chronic myocardial inflammation, oxidative and ER stress, and particles damaging myocardial organelles may prime the development of pathophysiological cardiovascular states in young urbanites. Nanosized PM could play a key cardiac myocyte toxicity role.

Up-Regulation of mRNA Ventricular PRNP Prion Protein Gene Expression in Air Pollution Highly Exposed Young Urbanites: Endoplasmic Reticulum Stress, Glucose Regulated Protein 78, and Nanosized Particles

PubMed Central

Villarreal-Calderon, Rodolfo; Franco-Lira, Maricela; González-Maciel, Angélica; Reynoso-Robles, Rafael; Harritt, Lou; Pérez-Guillé, Beatriz; Ferreira-Azevedo, Lara; Drecktrah, Dan; Zhu, Hongtu; Sun, Qiang; Torres-Jardón, Ricardo; Aragón-Flores, Mariana; Calderón-Garcidueñas, Ana; Diaz, Philippe; Calderón-Garcidueñas, Lilian

2013-01-01

Mexico City Metropolitan Area children and young adults exposed to high concentrations of air pollutants including fine and ultrafine particulate matter (PM) vs. clean air controls, exhibit myocardial inflammation and inflammasome activation with a differential right and left ventricular expression of key inflammatory genes and inflammasomes. We investigated the mRNA expression levels of the prion protein gene PRNP, which plays an important role in the protection against oxidative stress and metal toxicity, and the glucose regulated protein 78, a key protein in endoplasmic reticulum (ER) stress signaling, in ventricular autopsy samples from 30 children and young adults age 19.97 ± 6.8 years with a lifetime of low (n:4) vs. high (n:26) air pollution exposures. Light microscopy and transmission electron microscopy studies were carried out in human ventricles, and electron microscopy studies were also done in 5 young, highly exposed Mexico City dogs. There was significant left ventricular PRNP and bi-ventricular GRP78 mRNA up-regulation in Mexico City young urbanites vs. controls. PRNP up-regulation in the left ventricle was significantly different from the right, p < 0.0001, and there was a strong left ventricular PRNP and GRP78 correlation (p = 0.0005). Marked abnormalities in capillary endothelial cells, numerous nanosized particles in myocardial ER and in abnormal mitochondria characterized the highly exposed ventricles. Early and sustained cardiac ER stress could result in detrimental irreversible consequences in urban children, and while highly complex systems maintain myocardial homeostasis, failure to compensate for chronic myocardial inflammation, oxidative and ER stress, and particles damaging myocardial organelles may prime the development of pathophysiological cardiovascular states in young urbanites. Nanosized PM could play a key cardiac myocyte toxicity role. PMID:24287918

Codon optimisation to improve expression of a Mycobacterium avium ssp. paratuberculosis-specific membrane-associated antigen by Lactobacillus salivarius.

PubMed

Johnston, Christopher; Douarre, Pierre E; Soulimane, Tewfik; Pletzer, Daniel; Weingart, Helge; MacSharry, John; Coffey, Aidan; Sleator, Roy D; O'Mahony, Jim

2013-06-01

Subunit and DNA-based vaccines against Mycobacterium avium ssp. paratuberculosis (MAP) attempt to overcome inherent issues associated with whole-cell formulations. However, these vaccines can be hampered by poor expression of recombinant antigens from a number of disparate hosts. The high G+C content of MAP invariably leads to a codon bias throughout gene expression. To investigate if the codon bias affects recombinant MAP antigen expression, the open reading frame of a MAP-specific antigen MptD (MAP3733c) was codon optimised for expression against a Lactobacillus salivarius host. Of the total 209 codons which constitute MAP3733c, 172 were modified resulting in a reduced G+C content from 61% for the native gene to 32.7% for the modified form. Both genes were placed under the transcriptional control of the PnisA promoter; allowing controlled heterologous expression in L. salivarius. Expression was monitored using fluorescence microscopy and microplate fluorometry via GFP tags translationally fused to the C-termini of the two MptD genes. A > 37-fold increase in expression was observed for the codon-optimised MAP3733synth variant over the native gene. Due to the low cost and improved expression achieved, codon optimisation significantly improves the potential of L. salivarius as an oral vaccine stratagem against Johne's disease. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Verification of γ-Amino-Butyric Acid (GABA) Signaling System Components in Periodontal Ligament Cells In Vivo and In Vitro.

PubMed

Konermann, Anna; Kantarci, Alpdogan; Wilbert, Steven; Van Dyke, Thomas; Jäger, Andreas

2016-11-01

CNS key neurotransmitter γ-amino-butyric acid (GABA) and its signaling components are likewise detectable in non-neuronal tissues displaying inter alia immunomodulatory functions. This study aimed at identifying potential glutamate decarboxylase (GAD)65 and GABA receptor expression in periodontal ligament (PDL) cells in vivo and in vitro, with particular regard to inflammation and mechanical loading. Gene expression was analyzed in human PDL cells at rest or in response to IL-1ß (5 ng/ml) or TNFα (5 ng/ml) challenge via qRT-PCR. Western blot determined constitutive receptor expression, and confocal laser scanning fluorescence microscopy visualized expression changes induced by inflammation. ELISA quantified GAD65 release. Immunocytochemistry was performed for GABA component detection in vitro on mechanically loaded PDL cells, and in vivo on rat upper jaw biopsies with mechanically induced root resorptions. Statistical significance was set at p < 0.05. GABA B1 , GABA B2 , GABA A1 , and GABA A3 were ubiquitously expressed both on gene and protein level. GABA A2 and GAD65 were undetectable in resting cells, but induced by inflammation. GABA B1 exhibited the highest basal gene expression (6.97 % ± 0.16). IL-1ß markedly increased GABA B2 on a transcriptional (57.28-fold ± 12.40) and protein level seen via fluorescence microscopy. TNFα-stimulated PDL cells released GAD65 (3.68 pg/ml ± 0.17 after 24 h, 5.77 pg/ml ± 0.65 after 48 h). Immunocytochemistry revealed GAD65 expression in mechanically loaded PDL cells. In vivo, GABA components were varyingly expressed in an inflammatory periodontal environment. PDL cells differentially express GABA signaling components and secrete GAD65. Inflammation and mechanical loading regulate these neurotransmitter molecules, which are also detectable in vivo and are potentially involved in periodontal pathophysiology.

Analysis of the resistance mechanisms in sugarcane during Sporisorium scitamineum infection using RNA-seq and microscopy.

PubMed

McNeil, Meredith D; Bhuiyan, Shamsul A; Berkman, Paul J; Croft, Barry J; Aitken, Karen S

2018-01-01

Smut caused by biotrophic fungus Sporisorium scitamineum is a major disease of cultivated sugarcane that can cause considerable yield losses. It has been suggested in literature that there are at least two types of resistance mechanisms in sugarcane plants: an external resistance, due to chemical or physical barriers in the sugarcane bud, and an internal resistance governed by the interaction of plant and fungus within the plant tissue. Detailed molecular studies interrogating these two different resistance mechanisms in sugarcane are scarce. Here, we use light microscopy and global expression profiling with RNA-seq to investigate these mechanisms in sugarcane cultivar CP74-2005, a cultivar that possibly possesses both internal and external defence mechanisms. A total of 861 differentially expressed genes (DEGs) were identified in a comparison between infected and non-infected buds at 48 hours post-inoculation (hpi), with 457 (53%) genes successfully annotated using BLAST2GO software. This includes genes involved in the phenylpropanoid pathway, cell wall biosynthesis, plant hormone signal transduction and disease resistance genes. Finally, the expression of 13 DEGs with putative roles in S. scitamineum resistance were confirmed by quantitative real-time reverse transcription PCR (qRT-PCR) analysis, and the results were consistent with the RNA-seq data. These results highlight that the early sugarcane response to S. scitamineum infection is complex and many of the disease response genes are attenuated in sugarcane cultivar CP74-2005, while others, like genes involved in the phenylpropanoid pathway, are induced. This may point to the role of the different disease resistance mechanisms that operate in cultivars such as CP74-2005, whereby the early response is dominated by external mechanisms and then as the infection progresses, the internal mechanisms are switched on. Identification of genes underlying resistance in sugarcane will increase our knowledge of the sugarcane-S. scitamineum interaction and facilitate the introgression of new resistance genes into commercial sugarcane cultivars.

The concentration, gene expression, and spatial distribution of aggrecan in canine articular cartilage, meniscus, and anterior and posterior cruciate ligaments: a new molecular distinction between hyaline cartilage and fibrocartilage in the knee joint.

PubMed

Valiyaveettil, Manojkumar; Mort, John S; McDevitt, Cahir A

2005-01-01

The concentration, spatial distribution, and gene expression of aggrecan in meniscus, articular cartilage, and the anterior and posterior cruciate ligaments (ACL and PCL) was determined in the knee joints of five mature dogs. An anti-serum against peptide sequences specific to the G1 domain of aggrecan was employed in competitive-inhibition ELISA of guanidine HCl extracts and immunofluorescence microscopy. Gene expression was determined by Taqman real-time PCR. The concentration of aggrecan in articular cartilage (240.1 +/- 32 nMol/g dry weight) was higher than that in meniscus (medial meniscus: 33.4 +/- 4.3 nMol/g) and ligaments (ACL: 6.8 +/- 0.9 nMol/g). Aggrecan was more concentrated in the inner than the outer zone of the meniscus. Aggrecan in meniscus showed an organized, spatial network, in contrast to its diffuse distribution in articular cartilage. Thus, differences in the concentration, gene expression, and spatial distribution of aggrecan constitute another molecular distinction between hyaline cartilage and fibrocartilage of the knee.

Assessing the role of Pseudomonas aeruginosa surface-active gene expression in hexadecane biodegradation in sand.

PubMed

Holden, P A; LaMontagne, M G; Bruce, A K; Miller, W G; Lindow, S E

2002-05-01

Low pollutant substrate bioavailability limits hydrocarbon biodegradation in soils. Bacterially produced surface-active compounds, such as rhamnolipid biosurfactant and the PA bioemulsifying protein produced by Pseudomonas aeruginosa, can improve bioavailability and biodegradation in liquid culture, but their production and roles in soils are unknown. In this study, we asked if the genes for surface-active compounds are expressed in unsaturated porous media contaminated with hexadecane. Furthermore, if expression does occur, is biodegradation enhanced? To detect expression of genes for surface-active compounds, we fused the gfp reporter gene either to the promoter region of pra, which encodes for the emulsifying PA protein, or to the promoter of the transcriptional activator rhlR. We assessed green fluorescent protein (GFP) production conferred by these gene fusions in P. aeruginosa PG201. GFP was produced in sand culture, indicating that the rhlR and pra genes are both transcribed in unsaturated porous media. Confocal laser scanning microscopy of liquid drops revealed that gfp expression was localized at the hexadecane-water interface. Wild-type PG201 and its mutants that are deficient in either PA protein, rhamnolipid synthesis, or both were studied to determine if the genetic potential to make surface-active compounds confers an advantage to P. aeruginosa biodegrading hexadecane in sand. Hexadecane depletion rates and carbon utilization efficiency in sand culture were the same for wild-type and mutant strains, i.e., whether PG201 was proficient or deficient in surfactant or emulsifier production. Environmental scanning electron microscopy revealed that colonization of sand grains was sparse, with cells in small monolayer clusters instead of multilayered biofilms. Our findings suggest that P. aeruginosa likely produces surface-active compounds in sand culture. However, the ability to produce surface-active compounds did not enhance biodegradation in sand culture because well-distributed cells and well-distributed hexadecane favored direct contact to hexadecane for most cells. In contrast, surface-active compounds enable bacteria in liquid culture to adhere to the hexadecane-water interface when they otherwise would not, and thus production of surface-active compounds is an advantage for hexadecane biodegradation in well-dispersed liquid systems.

Intracellular processing of poly(ethylene imine)/ribozyme complexes can be observed in living cells by using confocal laser scanning microscopy and inhibitor experiments.

PubMed

Merdan, Thomas; Kunath, Klaus; Fischer, Dagmar; Kopecek, Jindrich; Kissel, Thomas

2002-02-01

Critical steps in the subcellular processing of poly(ethylene imine)/nucleic acid complexes, especially endosomal/lysosomal escape, were visualized by using living cell confocal laser scanning microscopy (CSLM) to obtain an insight into their mechanism. Living cell confocal microscopy was used to examine the intracellular fate of poly(ethylene imine)/ribozyme and poly(L-lysine)/ribozyme complexes over time, in the presence of and without bafilomycin Al, a selective inhibitor of endosomal/lysosomal acidification. The compartment of complex accumulation was identified by confocal microscopy with a fluorescent acidotropic dye. To confirm microscopic data, luciferase reporter gene expression was determined under similar experimental conditions. Poly(ethylene imine)/ribozyme complexes accumulate in acidic vesicles, most probably lysosomes. Release of complexes occurs in a sudden event, very likely due to bursting of these organelles. After release, poly(ethylene imine) and ribozyme spread throughout the cell, during which slight differences in distribution between cytosol and nucleus are visible. No lysosomal escape was observed with poly(L-lysine)/ribozyme complexes or when poly(ethylene imine)/ ribozyme complexes were applied together with bafilomycin A1. Poly(ethylene imine)/plasmid complexes exhibited a high luciferase expression, which was reduced approximately 200-fold when lysosomal acidification was suppressed with bafilomycin A1. Our data provide, for the first time, direct experimental evidence for the escape of poly(ethylene imine)/nucleic acid complexes from the endosomal/lysosomal compartment. CLSM, in conjunction with living cell microscopy, is a promising tool for studying the subcellular fate of polyplexes in nucleic acid/gene delivery.

Control of Flagellar Gene Regulation in Legionella pneumophila and Its Relation to Growth Phase▿ †

PubMed Central

Albert-Weissenberger, Christiane; Sahr, Tobias; Sismeiro, Odile; Hacker, Jörg; Heuner, Klaus; Buchrieser, Carmen

2010-01-01

The bacterial pathogen Legionella pneumophila responds to environmental changes by differentiation. At least two forms are well described: replicative bacteria are avirulent; in contrast, transmissive bacteria express virulence traits and flagella. Phenotypic analysis, Western blotting, and electron microscopy of mutants of the regulatory genes encoding RpoN, FleQ, FleR, and FliA demonstrated that flagellin expression is strongly repressed and that the mutants are nonflagellated in the transmissive phase. Transcriptome analyses elucidated that RpoN, together with FleQ, enhances transcription of 14 out of 31 flagellar class II genes, which code for the basal body, hook, and regulatory proteins. Unexpectedly, FleQ independent of RpoN enhances the transcription of fliA encoding sigma 28. Expression analysis of a fliA mutant showed that FliA activates three out of the five remaining flagellar class III genes and the flagellar class IV genes. Surprisingly, FleR does not induce but inhibits expression of at least 14 flagellar class III genes on the transcriptional level. Thus, we propose that flagellar class II genes are controlled by FleQ and RpoN, whereas the transcription of the class III gene fliA is controlled in a FleQ-dependent but RpoN-independent manner. However, RpoN and FleR might influence flagellin synthesis on a posttranscriptional level. In contrast to the commonly accepted view that enhancer-binding proteins such as FleQ always interact with RpoN to fullfill their regulatory functions, our results strongly indicate that FleQ regulates gene expression that is RpoN dependent and RpoN independent. Finally, FliA induces expression of flagellar class III and IV genes leading to the complete synthesis of the flagellum. PMID:19915024

Building quantitative, three-dimensional atlases of gene expression and morphology at cellular resolution.

PubMed

Knowles, David W; Biggin, Mark D

2013-01-01

Animals comprise dynamic three-dimensional arrays of cells that express gene products in intricate spatial and temporal patterns that determine cellular differentiation and morphogenesis. A rigorous understanding of these developmental processes requires automated methods that quantitatively record and analyze complex morphologies and their associated patterns of gene expression at cellular resolution. Here we summarize light microscopy-based approaches to establish permanent, quantitative datasets-atlases-that record this information. We focus on experiments that capture data for whole embryos or large areas of tissue in three dimensions, often at multiple time points. We compare and contrast the advantages and limitations of different methods and highlight some of the discoveries made. We emphasize the need for interdisciplinary collaborations and integrated experimental pipelines that link sample preparation, image acquisition, image analysis, database design, visualization, and quantitative analysis. Copyright © 2013 Wiley Periodicals, Inc.

Mycoparasitism studies of Trichoderma harzianum against Sclerotinia sclerotiorum: evaluation of antagonism and expression of cell wall-degrading enzymes genes.

PubMed

Troian, Rogério Fraga; Steindorff, Andrei Stecca; Ramada, Marcelo Henrique Soller; Arruda, Walquiria; Ulhoa, Cirano José

2014-10-01

Trichoderma spp. are known for their biocontrol activity against several plant pathogens. A specific isolate of Trichoderma harzianum, 303/02, has the potential to inhibit the growth of Sclerotinia sclerotiorum, an important agent involved in several crop diseases. In this study, the interaction between T. harzianum 303/02 and mycelia, sclerotia and apothecia of S. sclerotiorum was studied by scanning electron microscopy. RT-qPCR was used to examine the expression of 11 genes potentially involved in biocontrol. T. harzianum 303/02 parasitizes S. sclerotiorum by forming branches that coil around the hyphae. The fungus multiplied abundantly at the sclerotia and apothecia surface, forming a dense mycelium that penetrated the inner surface of these structures. The levels of gene expression varied according to the type of structure with which T. harzianum was interacting. The data also showed the presence of synergistic action between the cell-wall degrading enzymes.

Cardiomyogenic Differentiation in Cardiac Myxoma Expressing Lineage-Specific Transcription Factors

PubMed Central

Kodama, Hiroaki; Hirotani, Takashi; Suzuki, Yusuke; Ogawa, Satoshi; Yamazaki, Kazuto

2002-01-01

We investigated five cases of cardiac myxoma and one case of cardiac undifferentiated sarcoma by light and electron microscopy, in situ hybridization, immunohistochemical staining, and reverse transcriptase-polymerase chain reaction for cardiomyocyte-specific transcription factors, Nkx2.5/Csx, GATA-4, MEF2, and eHAND. Conventional light microscopy revealed that cardiac myxoma and sarcoma cells presented variable cellular arrangements and different histological characteristics. Ultrastructurally, some of the myxoma cells exhibited endothelium-like or immature mesenchymal cell differentiation. Immunohistochemistry for Nkx2.5/Csx, GATA-4, and eHAND was slightly to intensely positive in all myxoma cases. MEF2 immunoreactivity was observed in all cases including the case of sarcoma, thus suggesting myogenic differentiation of myxoma or sarcoma cells. In situ hybridization for Nkx2.5/Csx also revealed that all myxoma cells, but not sarcoma cells, expressed mRNA of the cardiac homeobox gene, Nkx2.5/Csx. Furthermore, nested reverse transcriptase-polymerase chain reaction from formalin-fixed, paraffin-embedded tissue was performed and demonstrated that the Nkx2.5/Csx and eHAND gene product to be detected in all cases, and in three of six cases, respectively. In conclusion, cardiac myxoma cells were found to express various amounts of cardiomyocyte-specific transcription factor gene products at the mRNA and protein levels, thus suggesting cardiomyogenic differentiation. These results support the concept that cardiac myxoma might arise from mesenchymal cardiomyocyte progenitor cells. PMID:12163362

Analysis of fiber-type differences in reporter gene expression of β-gal transgenic muscle.

PubMed

Tai, Phillip W L; Smith, Catherine L; Angello, John C; Hauschka, Stephen D

2012-01-01

β-galactosidase (β-gal) is among the most frequently used markers for studying a wide variety of biological mechanisms, e.g., gene expression, cell migration, stem cell conversion to different cell types, and gene silencing. Many of these studies require the histochemical detection of relative β-gal levels in tissue cross-sections mounted onto glass slides and visualized by microscopy. This is particularly useful for the analysis of promoter activity in skeletal muscle tissue since the β-gal levels can vary dramatically between different anatomical muscles and myofiber types. The differences in promoter activity can be due to a myofiber's developmental history, innervation, response to normal or experimental physiological signals, and its disease state. It is thus important to identify the individual fiber types within muscle cross-sections and to correlate these with transgene expression signals. Here, we provide a detailed description of how to process and analyze muscle tissues to determine the fiber-type composition and β-gal transgene expression within cryosections.

Non-plaque-forming virions of Modified Vaccinia virus Ankara express viral genes.

PubMed

Lülf, Anna-Theresa; Freudenstein, Astrid; Marr, Lisa; Sutter, Gerd; Volz, Asisa

2016-12-01

In cell culture infections with vaccinia virus the number of counted virus particles is substantially higher than the number of plaques obtained by titration. We found that standard vaccine preparations of recombinant Modified Vaccinia virus Ankara produce only about 20-30% plaque-forming virions in fully permissive cell cultures. To evaluate the biological activity of the non-plaque-forming particles, we generated recombinant viruses expressing fluorescent reporter proteins under transcriptional control of specific viral early and late promoters. Live cell imaging and automated counting by fluorescent microscopy indicated that virtually all virus particles can enter cells and switch on viral gene expression. Although most of the non-plaque-forming infections are arrested at the level of viral early gene expression, we detected activation of late viral transcription in 10-20% of single infected cells. Thus, non-plaque-forming particles are biologically active, and likely contribute to the immunogenicity of vaccinia virus vaccines. Copyright © 2016 Elsevier Inc. All rights reserved.

Development and characterization of a eukaryotic expression system for human type II procollagen.

PubMed

Wieczorek, Andrew; Rezaei, Naghmeh; Chan, Clara K; Xu, Chuan; Panwar, Preety; Brömme, Dieter; Merschrod S, Erika F; Forde, Nancy R

2015-12-15

Triple helical collagens are the most abundant structural protein in vertebrates and are widely used as biomaterials for a variety of applications including drug delivery and cellular and tissue engineering. In these applications, the mechanics of this hierarchically structured protein play a key role, as does its chemical composition. To facilitate investigation into how gene mutations of collagen lead to disease as well as the rational development of tunable mechanical and chemical properties of this full-length protein, production of recombinant expressed protein is required. Here, we present a human type II procollagen expression system that produces full-length procollagen utilizing a previously characterized human fibrosarcoma cell line for production. The system exploits a non-covalently linked fluorescence readout for gene expression to facilitate screening of cell lines. Biochemical and biophysical characterization of the secreted, purified protein are used to demonstrate the proper formation and function of the protein. Assays to demonstrate fidelity include proteolytic digestion, mass spectrometric sequence and posttranslational composition analysis, circular dichroism spectroscopy, single-molecule stretching with optical tweezers, atomic-force microscopy imaging of fibril assembly, and transmission electron microscopy imaging of self-assembled fibrils. Using a mammalian expression system, we produced full-length recombinant human type II procollagen. The integrity of the collagen preparation was verified by various structural and degradation assays. This system provides a platform from which to explore new directions in collagen manipulation.

Low RNA translation activit limits the efficacy of hydrodynamic gene transfer to pig liver in vivo.

PubMed

Sendra, Luis; Carreño, Omar; Miguel, Antonio; Montalvá, Eva; Herrero, María José; Orbis, Francisco; Noguera, Inmaculada; Barettino, Domingo; López-Andújar, Rafael; Aliño, Salvador F

2014-01-01

Hydrodynamic gene delivery has proved an efficient strategy for nonviral gene therapy in the murine liver but it has been less efficient in pigs. The reason for such inefficiency remains unclear. The present study used a surgical strategy to seal the whole pig liver in vivo. A solution of enhanced green fluorescent protein (eGFP) DNA was injected under two different venous injection conditions (anterograde and retrograde), employing flow rates of 10 and 20 ml/s in each case, with the aim of identifying the best gene transfer conditions. The gene delivery and information decoding steps were evaluated by measuring the eGFP DNA, mRNA and protein copy number 24 h after transfection. In addition, gold nanoparticles (diameters of 4 and 15 nm) were retrogradely injected (10 ml/s) to observe, by electron microscopy, the ability of the particle to access the hepatocyte. The gene delivery level was higher with anterograde injection, whereas the efficacy of gene expression was better with retrograde injection, suggesting differences in the decoding processes. Thus, retrograde injection mediates gene transcription (mRNA copy/cell) equivalent to that of intermediate expression proteins but the mRNA translation was lower than that of rare proteins. Electron microscopy showed that nanoparticles within the hepatocyte were almost exclusively 4 nm in diameter. The results suggest that the low activity of mRNA translation limits the final efficacy of the gene transfer procedure. On the other hand, the gold nanoparticles study suggests that elongated DNA conformation could offer advantages in that the access of 15-nm particles is very limited. Copyright © 2014 John Wiley & Sons, Ltd.

A homeodomain transcription factor gene, PfMSX, activates expression of Pif gene in the pearl oyster Pinctada fucata.

PubMed

Zhao, Mi; He, Maoxian; Huang, Xiande; Wang, Qi

2014-01-01

We reported pearl oyster Pinctada fucata cDNA and genomic characterization of a new homeobox-containing protein, PfMSX. The PfMSX gene encodes a transcription factor that was localized to the nucleus. Analyses of PfMSX mRNA in tissues and developmental stages showed high expressions in mantle or D-shaped larvae. In electrophoretic mobility shift assays (EMSAs) PfMSX binded to MSX consensus binding sites in the 5' flanking region of the Pif promoter. In co-transfection experiment PfMSX transactivated reporter constructs containing Pif promoter sequences, and mutation of the MSX-binding sites attenuated transactivation. A knockdown experiment using PfMSX dsRNA showed decreased Pif mRNA and unregular crystallization of the nacreous layer using scanning electron microscopy. Our results suggested that PfMSX was a conserved homeodomain transcription factor gene, which can activate Pif gene expression through MSX binding site, and was then involved in the mineralization process in pearl oyster Pinctada fucata. Our data provided important clues about mechanisms regulating biomineralization in pearl oyster.

A Homeodomain Transcription Factor Gene, PfMSX, Activates Expression of Pif Gene in the Pearl Oyster Pinctada fucata

PubMed Central

Zhao, Mi; He, Maoxian; Huang, Xiande; Wang, Qi

2014-01-01

We reported pearl oyster Pinctada fucata cDNA and genomic characterization of a new homeobox-containing protein, PfMSX. The PfMSX gene encodes a transcription factor that was localized to the nucleus. Analyses of PfMSX mRNA in tissues and developmental stages showed high expressions in mantle or D-shaped larvae. In electrophoretic mobility shift assays (EMSAs) PfMSX binded to MSX consensus binding sites in the 5′ flanking region of the Pif promoter. In co-transfection experiment PfMSX transactivated reporter constructs containing Pif promoter sequences, and mutation of the MSX-binding sites attenuated transactivation. A knockdown experiment using PfMSX dsRNA showed decreased Pif mRNA and unregular crystallization of the nacreous layer using scanning electron microscopy. Our results suggested that PfMSX was a conserved homeodomain transcription factor gene, which can activate Pif gene expression through MSX binding site, and was then involved in the mineralization process in pearl oyster Pinctada fucata. Our data provided important clues about mechanisms regulating biomineralization in pearl oyster. PMID:25099698

Expression of the Bovine NK-Lysin Gene Family and Activity against Respiratory Pathogens.

PubMed

Chen, Junfeng; Yang, Chingyuan; Tizioto, Polyana C; Huang, Huan; Lee, Mi O K; Payne, Harold R; Lawhon, Sara D; Schroeder, Friedhelm; Taylor, Jeremy F; Womack, James E

2016-01-01

Unlike the genomes of many mammals that have a single NK-lysin gene, the cattle genome contains a family of four genes, one of which is expressed preferentially in the lung. In this study, we compared the expression of the four bovine NK-lysin genes in healthy animals to animals challenged with pathogens known to be associated with bovine respiratory disease (BRD) using transcriptome sequencing (RNA-seq). The expression of several NK-lysins, especially NK2C, was elevated in challenged relative to control animals. The effects of synthetic peptides corresponding to functional region helices 2 and 3 of each gene product were tested on both model membranes and bio-membranes. Circular dichroism spectroscopy indicated that these peptides adopted a more helical secondary structure upon binding to an anionic model membrane and liposome leakage assays suggested that these peptides disrupt membranes. Bacterial killing assays further confirmed the antimicrobial effects of these peptides on BRD-associated bacteria, including both Pasteurella multocida and Mannhemia haemolytica and an ultrastructural examination of NK-lysin-treated P. multocida cells by transmission electron microscopy revealed the lysis of target membranes. These studies demonstrate that the expanded bovine NK-lysin gene family is potentially important in host defense against pathogens involved in bovine respiratory disease.

Expression of the human hepatitis B virus large surface antigen gene in transgenic tomato plants.

PubMed

Lou, Xiao-Ming; Yao, Quan-Hong; Zhang, Zhen; Peng, Ri-He; Xiong, Ai-Sheng; Wang, Hua-Kun

2007-04-01

The original hepatitis B virus (HBV) large surface antigen gene was synthesized. In order to optimize the expression of this gene in tomato plants, the tobacco pathogenesis-related protein S signal peptide was fused to the 5' end of the modified gene and the sequence encoding amino acids S, E, K, D, E, and L was placed at the 3' end. The gene encoding the modified HBV large surface antigen under the control of a fruit-specific promoter was constructed and expressed in transgenic tomato plants. The expression of the antigen from transgenic plants was confirmed by PCR and reverse transcriptase PCR. Enzyme-linked immunoassays using a monoclonal antibody directed against human serum-derived HBsAg revealed that the maximal level of HBsAg was about 0.02% of the soluble protein in transgenic tomato fruit. The amount of HBsAg in mature fruits was found to be 65- to 171-fold larger than in small or medium fruits and leaf tissues. Examination of transgenic plant samples by transmission electron microscopy proved that HBsAg had been expressed and had accumulated. The HBsAg protein was capable of assembling into capsomers and virus-like particles. To our knowledge, this is the first time the HBV large surface antigen has been expressed in plants. This work suggests the possibility of producing a new alternative vaccine for human HBV.

RNA-seq Analysis of Host and Viral Gene Expression Highlights Interaction between Varicella Zoster Virus and Keratinocyte Differentiation

PubMed Central

Singh, Manuraj; Kanda, Ravinder K.; Yee, Michael B.; Kellam, Paul; Hollinshead, Michael; Kinchington, Paul R.; O'Toole, Edel A.; Breuer, Judith

2014-01-01

Varicella zoster virus (VZV) is the etiological agent of chickenpox and shingles, diseases characterized by epidermal skin blistering. Using a calcium-induced keratinocyte differentiation model we investigated the interaction between epidermal differentiation and VZV infection. RNA-seq analysis showed that VZV infection has a profound effect on differentiating keratinocytes, altering the normal process of epidermal gene expression to generate a signature that resembles patterns of gene expression seen in both heritable and acquired skin-blistering disorders. Further investigation by real-time PCR, protein analysis and electron microscopy revealed that VZV specifically reduced expression of specific suprabasal cytokeratins and desmosomal proteins, leading to disruption of epidermal structure and function. These changes were accompanied by an upregulation of kallikreins and serine proteases. Taken together VZV infection promotes blistering and desquamation of the epidermis, both of which are necessary to the viral spread and pathogenesis. At the same time, analysis of the viral transcriptome provided evidence that VZV gene expression was significantly increased following calcium treatment of keratinocytes. Using reporter viruses and immunohistochemistry we confirmed that VZV gene and protein expression in skin is linked with cellular differentiation. These studies highlight the intimate host-pathogen interaction following VZV infection of skin and provide insight into the mechanisms by which VZV remodels the epidermal environment to promote its own replication and spread. PMID:24497829

Non-Viral Transfection Methods Optimized for Gene Delivery to a Lung Cancer Cell Line

PubMed Central

Salimzadeh, Loghman; Jaberipour, Mansooreh; Hosseini, Ahmad; Ghaderi, Abbas

2013-01-01

Background Mehr-80 is a newly established adherent human large cell lung cancer cell line that has not been transfected until now. This study aims to define the optimal transfection conditions and effects of some critical elements for enhancing gene delivery to this cell line by utilizing different non-viral transfection Procedures. Methods In the current study, calcium phosphate (CaP), DEAE-dextran, superfect, electroporation and lipofection transfection methods were used to optimize delivery of a plasmid construct that expressed Green Fluorescent Protein (GFP). Transgene expression was detected by fluorescent microscopy and flowcytometry. Toxicities of the methods were estimated by trypan blue staining. In order to evaluate the density of the transfected gene, we used a plasmid construct that expressed the Stromal cell-Derived Factor-1 (SDF-1) gene and measured its expression by real-time PCR. Results Mean levels of GFP-expressing cells 48 hr after transfection were 8.4% (CaP), 8.2% (DEAE-dextran), 4.9% (superfect), 34.1% (electroporation), and 40.1% (lipofection). Lipofection had the highest intense SDF-1 expression of the analyzed methods. Conclusion This study has shown that the lipofection and electroporation methods were more efficient at gene delivery to Mehr-80 cells. The quantity of DNA per transfection, reagent concentration, and incubation time were identified as essential factors for successful transfection in all of the studied methods. PMID:23799175

Pulmonary instillation of low doses of titanium dioxide nanoparticles in mice leads to particle retention and gene expression changes in the absence of inflammation

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

Husain, Mainul, E-mail: mainul.husain@hc-sc.gc.ca; Saber, Anne T., E-mail: ats@nrcwe.dk; Guo, Charles, E-mail: charles.guo@hc-sc.gc.ca

2013-06-15

We investigated gene expression, protein synthesis, and particle retention in mouse lungs following intratracheal instillation of varying doses of nano-sized titanium dioxide (nano-TiO{sub 2}). Female C57BL/6 mice were exposed to rutile nano-TiO{sub 2} via single intratracheal instillations of 18, 54, and 162 μg/mouse. Mice were sampled 1, 3, and 28 days post-exposure. The deposition of nano-TiO{sub 2} in the lungs was assessed using nanoscale hyperspectral microscopy. Biological responses in the pulmonary system were analyzed using DNA microarrays, pathway-specific real-time RT-PCR (qPCR), gene-specific qPCR arrays, and tissue protein ELISA. Hyperspectral mapping showed dose-dependent retention of nano-TiO{sub 2} in the lungs upmore » to 28 days post-instillation. DNA microarray analysis revealed approximately 3000 genes that were altered across all treatment groups (± 1.3 fold; p < 0.1). Several inflammatory mediators changed in a dose- and time-dependent manner at both the mRNA and protein level. Although no influx of neutrophils was detected at the low dose, changes in the expression of several genes and proteins associated with inflammation were observed. Resolving inflammation at the medium dose, and lack of neutrophil influx in the lung fluid at the low dose, were associated with down-regulation of genes involved in ion homeostasis and muscle regulation. Our gene expression results imply that retention of nano-TiO{sub 2} in the absence of inflammation over time may potentially perturb calcium and ion homeostasis, and affect smooth muscle activities. - Highlights: • Pulmonary effects following exposure to low doses of nano-TiO{sub 2} were examined. • Particle retention in lungs was assessed using nanoscale hyperspectral microscopy. • Particles persisted up to 28 days in lungs in all dose groups. • Inflammation was the pathway affected in the high dose group at all time points. • Ion homeostasis and muscle activity pathways were affected in the low dose group.« less

Expression loss and revivification of RhoB gene in ovary carcinoma carcinogenesis and development.

PubMed

Liu, Yingwei; Song, Na; Ren, Kexing; Meng, Shenglan; Xie, Yao; Long, Qida; Chen, Xiancheng; Zhao, Xia

2013-01-01

RhoB, a member of small GTPases belonging to the Ras protein superfamily, might have a suppressive activity in cancer progression. Here, expression of RhoB gene was evaluated in human benign, borderline and malignant ovary tumors by immunostaining, with normal ovary tissue as control. Malignant tumors were assessed according to Federation Internationale de Gynecologie Obstetrique (FIGO) guidelines and classified in stage I-IV. Revivification of RhoB gene was investigated by analyzing the effect of histone deacetylase (HDAC) inhibitor trichostatin (TSA) and methyltransferase inhibitor 5-azacytidine (5-Aza) on ovarian cancer cells via RT-PCR and western blot. Apoptosis of ovary cancer cells was detected using flowcytometry and fluorescence microscopy. Subsequently, RhoB expression is detected in normal ovary epithelium, borderline tumors, and decreases significantly or lost in the majority of ovarian cancer specimen (P<0.05). RhoB expression decreases significantly from stage II (71.4%) to stage III (43.5%) to stage IV (18.2%, P<0.05). TSA can both significantly revive the RhoB gene and mediate apoptosis of ovarian cancer cells, but 5-Aza couldn't. Interference into Revivification of RhoB gene results in reduction of ovary carcinoma cell apoptosis. It is proposed that loss of RhoB expression occurs frequently in ovary carcinogenesis and progression and its expression could be regulated by histone deacetylation but not by promoter hypermethylation, which may serve as a prospective gene treatment target for the patients with ovarian malignancy not responding to standard therapies.

Expression Loss and Revivification of RhoB Gene in Ovary Carcinoma Carcinogenesis and Development

PubMed Central

Liu, Yingwei; Song, Na; Ren, Kexing; Meng, Shenglan; Xie, Yao; Long, Qida; Chen, Xiancheng; Zhao, Xia

2013-01-01

RhoB, a member of small GTPases belonging to the Ras protein superfamily, might have a suppressive activity in cancer progression. Here, expression of RhoB gene was evaluated in human benign, borderline and malignant ovary tumors by immunostaining, with normal ovary tissue as control. Malignant tumors were assessed according to Federation Internationale de Gynecologie Obstetrique (FIGO) guidelines and classified in stage I-IV. Revivification of RhoB gene was investigated by analyzing the effect of histone deacetylase (HDAC) inhibitor trichostatin (TSA) and methyltransferase inhibitor 5-azacytidine (5-Aza) on ovarian cancer cells via RT-PCR and western blot. Apoptosis of ovary cancer cells was detected using flowcytometry and fluorescence microscopy. Subsequently, RhoB expression is detected in normal ovary epithelium, borderline tumors, and decreases significantly or lost in the majority of ovarian cancer specimen (P<0.05). RhoB expression decreases significantly from stage II (71.4%) to stage III (43.5%) to stage IV (18.2%, P<0.05). TSA can both significantly revive the RhoB gene and mediate apoptosis of ovarian cancer cells, but 5-Aza couldn’t. Interference into Revivification of RhoB gene results in reduction of ovary carcinoma cell apoptosis. It is proposed that loss of RhoB expression occurs frequently in ovary carcinogenesis and progression and its expression could be regulated by histone deacetylation but not by promoter hypermethylation, which may serve as a prospective gene treatment target for the patients with ovarian malignancy not responding to standard therapies. PMID:24223801

Physiological and molecular responses of Lactuca sativa to colonization by Salmonella enterica serovar Dublin.

PubMed

Klerks, M M; van Gent-Pelzer, M; Franz, E; Zijlstra, C; van Bruggen, A H C

2007-08-01

This paper describes the physiological and molecular interactions between the human-pathogenic organism Salmonella enterica serovar Dublin and the commercially available mini Roman lettuce cv. Tamburo. The association of S. enterica serovar Dublin with lettuce plants was first determined, which indicated the presence of significant populations outside and inside the plants. The latter was evidenced from significant residual concentrations after highly efficient surface disinfection (99.81%) and fluorescence microscopy of S. enterica serovar Dublin in cross sections of lettuce at the root-shoot transition region. The plant biomass was reduced significantly compared to that of noncolonized plants upon colonization with S. enterica serovar Dublin. In addition to the physiological response, transcriptome analysis by cDNA amplified fragment length polymorphism analysis also provided clear differential gene expression profiles between noncolonized and colonized lettuce plants. From these, generally and differentially expressed genes were selected and identified by sequence analysis, followed by reverse transcription-PCR displaying the specific gene expression profiles in time. Functional grouping of the expressed genes indicated a correlation between colonization of the plants and an increase in expressed pathogenicity-related genes. This study indicates that lettuce plants respond to the presence of S. enterica serovar Dublin at physiological and molecular levels, as shown by the reduction in growth and the concurrent expression of pathogenicity-related genes. In addition, it was confirmed that Salmonella spp. can colonize the interior of lettuce plants, thus potentially imposing a human health risk when processed and consumed.

[Ursodeoxycholic acid induced apoptosis of human hepatoma cells HepG2 and SMMC-7721 bymitochondrial-mediated pathway].

PubMed

Wu, Duan; Zhou, Jianyin; Yin, Zhenyu; Liu, Pingguo; Zhao, Yilin; Liu, Jianming; Wang, Xiaomin

2014-12-02

To explore the effects and underlying mechanisms of ursodeoxycholic acid on human hepatoma cells. HepG2 and SMMC-7721 HCC cell lines were respectively treated with ursodeoxycholic acid. And cell proliferation, apoptosis and the expression of Bax/Bcl-2 gene were detected by methyl thiazolyl tetrazolium (MTT), inverted microscopy, fluorescent microscopy, flow cytometry and Western blot. Ursodeoxycholic acid significantly inhibited the proliferation of human hepatoma cells in a concentration- and time-dependent manner. The half maximal inhibitory concentrations (IC50) of HepG2 and SMMC-7721 were 397.3 and 387.7 µg/ml respectively after a 48-hour treatment of 400 µg /ml ursodeoxycholic acid. And it also induced the apoptosis of HepG2 and SMMC-7721 cells, up-regulated Bax gene and down-regulated Bcl-2 gene. Ursodeoxycholic acid inhibits the proliferation of hepatoma cells and induce apoptosis by mitochondrial-mediated pathway.

Loss of notochordal cell phenotype in 3D-cell cultures: implications for disc physiology and disc repair.

PubMed

Omlor, G W; Nerlich, A G; Tirlapur, U K; Urban, J P; Guehring, T

2014-12-01

Embryonic notochordal disc nucleus cells (NC) have been identified to protect disc tissue against disc degeneration but in human beings NC phenotype gets lost with aging and the pathophysiological mechanisms are poorly understood. NC may stimulate other cells via soluble factors, and NC-conditioned medium can be used to stimulate matrix production of other disc cells and mesenchymal stem cells and thus may be of special interest for biological disc repair. As this stimulatory effect is associated with the NC phenotype, we investigated how cell morphology and gene-expression of the NC phenotype changes with time in 3D-cell culture. NC and inner annulus chondrocyte-like cells (CLC) from immature pigtails (freshly isolated cells/tissue, 3D-alginate beads, 3D-clusters) were cultured for up to 16 days under normoxia and hypoxia. Protein-expression was analysed by immunohistology and gene-expression analysis was carried out on freshly isolated cells and cultured cells. Cell morphology and proliferation were analysed by two-photon-laser-microscopy. Two-photon-laser-microscopy showed a homogenous and small CLC population in the inner annulus, which differed from the large vacuole-containing NC in the nucleus. Immunohistology found 93 % KRT8 positive cells in the nucleus and intracellular and pericellular Col2, IL6, and IL12 staining while CLC were KRT8 negative. Freshly isolated NC showed significantly higher KRT8 and CAIII but lower Col2 gene-expression than CLC. NC in 3D-cultures demonstrated significant size reduction and loss of vacuoles with culture time, all indicating a loss of the characteristic NC morphology. Hypoxia reduced the rate of decrease in NC size and vacuoles. Gene-expression of KRT8 and CAIII in NC fell significantly early in culture while Col2 did not decrease significantly within the culture period. In CLC, KRT8 and CAIII gene-expression was low and did not change noticeably in culture, whereas Col2 expression fell with time in culture. 3D-culture caused a rapid loss of NC phenotype towards a CLC phenotype with disappearance of vacuoles, reduced cell size, increased proliferation, and gene-expression changes. These findings may be related to NC nutritional demands and support the latest hypothesis of NC maturation into CLC opposing the idea that NC get lost in human discs by cell death or apoptosis to be replaced by CLC from the inner annulus.

Functional characterization of a novel jasmonate ZIM-domain interactor (NINJA) from upland cotton (Gossypium hirsutum).

PubMed

Wang, Le; Wu, Shu-Ming; Zhu, Yue; Fan, Qiang; Zhang, Zhen-Nan; Hu, Guang; Peng, Qing-Zhong; Wu, Jia-He

2017-03-01

The jasmonic acid (JA) signalling pathway plays roles in plant development and defence against biotic and abiotic stresses. We isolated a cotton NINJA (novel interactor of JA ZIM-domain) gene, designated GhNINJA, which contains a 1305 bp open read frame. The GhNINJA gene encodes a 434 amino acid peptide. According to quantitative real-time PCR analysis, GhNINJA is preferentially expressed in roots, and its expression level is greatly induced by Verticillium dahliae infection. Through a virus-induced gene silencing technique, we developed GhNINJA-silenced cotton plants, which had significantly decreased expression of the target gene with an average expression of 6% of the control. The regenerating lateral root growth of silenced plants was largely inhibited compared to the control. Analysis by microscopy demonstrated that the cell length of the root differentiation zone in GhNINJA-silenced plants is significantly shorter than those of the control. Moreover, the silenced plants exhibited higher tolerance to V. dahliae infection compared to the control, which was linked to the increased expression of the defence marker genes PDF1.2 and PR4. Together, these data indicated that knockdown of GhNINJA represses the root growth and enhances the tolerance to V. dahliae. Therefore, GhNINJA gene can be used as a candidate gene to breed the new cultivars for improving cotton yield and disease resistance. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Clausa, a Tomato Mutant with a Wide Range of Phenotypic Perturbations, Displays a Cell Type-Dependent Expression of the Homeobox Gene LeT6/TKn21

PubMed Central

Avivi, Yigal; Lev-Yadun, Simcha; Morozova, Nadya; Libs, Laurence; Williams, Leor; Zhao, Jing; Varghese, George; Grafi, Gideon

2000-01-01

Class I knox genes play an important role in shoot meristem function and are thus involved in the ordered development of stems, leaves, and reproductive organs. To elucidate the mechanism underlying the expression pattern of these homeobox genes, we studied a spontaneous tomato (Lycopersicon esculentum) mutant that phenotypically resembles, though is more extreme than, transgenic plants misexpressing class I knox genes. This mutant was found to carry a recessive allele, denoted clausa:shootyleaf (clau:shl)—a newly identified allele of clausa. Mutant plants exhibited abnormal leaf and flower morphology, epiphyllus inflorescences, fusion of organs, calyx asymmetry, and navel-like fruits. Analysis by scanning electron microscopy revealed that such fruits carried ectopic ovules, various vegetative primordia, as well as “forests” of stalked glandular trichomes. In situ RNA hybridization showed a peculiar expression pattern of the class I knox gene LeT6/TKn2; expression was restricted to the vascular system and palisade layer of mature leaves and to the inner part of ovules integuments. We conclude that CLAUSA regulates various aspects of tomato plant development, at least partly, by rendering the LeT6/TKn2 gene silent in specific tissues during development. Considering the expression pattern of LeT6/TKn2 in the clausa mutant, we suggest that the control over a given homeobox gene is maintained by several different regulatory mechanisms, in a cell type-dependent manner. PMID:11027705

Mapping the co-localization of the circadian proteins PER2 and BMAL1 with enkephalin and substance P throughout the rodent forebrain.

PubMed

Frederick, Ariana; Goldsmith, Jory; de Zavalia, Nuria; Amir, Shimon

2017-01-01

Despite rhythmic expression of clock genes being found throughout the central nervous system, very little is known about their function outside of the suprachiasmatic nucleus. Determining the pattern of clock gene expression across neuronal subpopulations is a key step in understanding their regulation and how they may influence the functions of various brain structures. Using immunofluorescence and confocal microscopy, we quantified the co-expression of the clock proteins BMAL1 and PER2 with two neuropeptides, Substance P (SubP) and Enkephalin (Enk), expressed in distinct neuronal populations throughout the forebrain. Regions examined included the limbic forebrain (dorsal striatum, nucleus accumbens, amygdala, stria terminalis), thalamus medial habenula of the thalamus, paraventricular nucleus and arcuate nucleus of the hypothalamus and the olfactory bulb. In most regions examined, BMAL1 was homogeneously expressed in nearly all neurons (~90%), and PER2 was expressed in a slightly lower proportion of cells. There was no specific correlation to SubP- or Enk- expressing subpopulations. The olfactory bulb was unique in that PER2 and BMAL1 were expressed in a much smaller percentage of cells, and Enk was rarely found in the same cells that expressed the clock proteins (SubP was undetectable). These results indicate that clock genes are not unique to specific cell types, and further studies will be required to determine the factors that contribute to the regulation of clock gene expression throughout the brain.

Long-depth imaging of specific gene expressions in whole-mount mouse embryos with single-photon excitation confocal fluorescence microscopy and FISH.

PubMed

Palmes-Saloma, C; Saloma, C

2000-07-01

Long-depth imaging of specific gene expression in the midgestation whole-mount mouse embryo (WME) is demonstrated with single-photon excitation (1PE) confocal fluorescence microscopy and fluorescence in situ hybridization. Expression domains of Pax-6 mRNA transcripts were labeled with an in situ hybridization probe that is a RNA sequence complementary to the cloned gene fragment and were rendered visible using two fluorochrome-conjugated antibodies that fluoresce at peak wavelengths of lambda(F) = 0.525 microm and lambda(F) = 0. 580 microm, respectively. Distributions of Pax-6 mRNA domains as deep as 1000 microm in the day 9.5 WME were imaged with a long-working-distance (13.6 mm) objective lens (magnification 5x). The scattering problem posed by the optically thick WME sample is alleviated by careful control of the detector pinhole size and the application of simple but fast postdetection image enhancement techniques, such as space and wavelength averaging to produce high-quality fluorescence images. A three-dimensional reconstruction that clearly shows the Pax-6 mRNA expression domains in the forebrain, diencephalon, optic cup, and spinal cord of the day 9.5 WME is obtained. The advantages of 1PE confocal fluorescence imaging over two-photon excitation fluorescence imaging are discussed for the case of long-depth imaging in highly scattering media. Imaging in midgestation WMEs at optical depths of more than 350 microm has not yet been realized with two-photon fluorescence excitation. Copyright 2000 Academic Press.

Radioluminescence Microscopy: Measuring the Heterogeneous Uptake of Radiotracers in Single Living Cells

PubMed Central

Pratx, Guillem; Chen, Kai; Sun, Conroy; Martin, Lynn; Carpenter, Colin M.; Olcott, Peter D.; Xing, Lei

2012-01-01

Radiotracers play an important role in interrogating molecular processes both in vitro and in vivo. However, current methods are limited to measuring average radiotracer uptake in large cell populations and, as a result, lack the ability to quantify cell-to-cell variations. Here we apply a new technique, termed radioluminescence microscopy, to visualize radiotracer uptake in single living cells, in a standard fluorescence microscopy environment. In this technique, live cells are cultured sparsely on a thin scintillator plate and incubated with a radiotracer. Light produced following beta decay is measured using a highly sensitive microscope. Radioluminescence microscopy revealed strong heterogeneity in the uptake of [18F]fluoro-deoxyglucose (FDG) in single cells, which was found consistent with fluorescence imaging of a glucose analog. We also verified that dynamic uptake of FDG in single cells followed the standard two-tissue compartmental model. Last, we transfected cells with a fusion PET/fluorescence reporter gene and found that uptake of FHBG (a PET radiotracer for transgene expression) coincided with expression of the fluorescent protein. Together, these results indicate that radioluminescence microscopy can visualize radiotracer uptake with single-cell resolution, which may find a use in the precise characterization of radiotracers. PMID:23056276

The quest for four-dimensional imaging in plant cell biology: it's just a matter of time

PubMed Central

Domozych, David S.

2012-01-01

Background Analysis of plant cell dynamics over time, or four-dimensional imaging (4-DI), represents a major goal of plant science. The ability to resolve structures in the third dimension within the cell or tissue during developmental events or in response to environmental or experimental stresses (i.e. 4-DI) is critical to our understanding of gene expression, post-expression modulations of macromolecules and sub-cellular system interactions. Scope Microscopy-based technologies have been profoundly integral to this type of investigation, and new and refined microscopy technologies now allow for the visualization of cell dynamics with unprecedented resolution, contrast and experimental versatility. However, certain realities of light and electron microscopy, choice of specimen and specimen preparation techniques limit the scope of readily attaining 4-DI. Today, the plant microscopist must use a combinatorial strategy whereby multiple microscopy-based investigations are used. Modern fluorescence, confocal laser scanning, transmission electron and scanning electron microscopy provide effective conduits for synthesizing data detailing live cell dynamics and highly resolved snapshots of specific cell structures that will ultimately lead to 4-DI. This review provides a synopsis of such technologies available. PMID:22628381

Microarray analysis of the rat lacrimal gland following the loss of parasympathetic control of secretion

PubMed Central

Nguyen, Doan H.; Toshida, Hiroshi; Schurr, Jill; Beuerman, Roger W.

2010-01-01

Previous studies showed that loss of muscarinic parasympathetic input to the lacrimal gland (LG) leads to a dramatic reduction in tear secretion and profound changes to LG structure. In this study, we used DNA microarrays to examine the regulation of the gene expression of the genes for secretory function and organization of the LG. Long-Evans rats anesthetized with a mixture of ketamine/xylazine (80:10 mg/kg) underwent unilateral sectioning of the greater superficial petrosal nerve, the input to the pterygopalatine ganglion. After 7 days, tear secretion was measured, the animals were killed, and structural changes in the LG were examined by light microscopy. Total RNA from control and experimental LGs (n = 5) was used for DNA microarray analysis employing the U34A GeneChip. Three statistical algorithms (detection, change call, and signal log ratio) were used to determine differential gene expression using the Microarray Suite (5.0) and Data Mining Tools (3.0). Tear secretion was significantly reduced and corneal ulcers developed in all experimental eyes. Light microscopy showed breakdown of the acinar structure of the LG. DNA microarray analysis showed downregulation of genes associated with the endoplasmic reticulum and Golgi, including genes involved in protein folding and processing. Conversely, transcripts for cytoskeleton and extracellular matrix components, inflammation, and apoptosis were upregulated. The number of significantly upregulated genes (116) was substantially greater than the number of downregulated genes (49). Removal of the main secretory input to the rat LG resulted in clinical symptoms associated with severe dry eye. Components of the secretory pathway were negatively affected, and the increase in cell proliferation and inflammation may lead to loss of organization in the parasympathectomized lacrimal gland. PMID:15084711

Synthesis and characterization of polyamidoamine dendrimer-coated multi-walled carbon nanotubes and their application in gene delivery systems

NASA Astrophysics Data System (ADS)

Pan, Bifeng; Cui, Daxiang; Xu, Ping; Ozkan, Cengiz; Feng, Gao; Ozkan, Mihri; Huang, Tuo; Chu, Bingfeng; Li, Qing; He, Rong; Hu, Guohan

2009-03-01

With the aim of improving the amount and delivery efficiency of genes taken by carbon nanotubes into human cancer cells, different generations of polyamidoamine dendrimer modified multi-walled carbon nanotubes (dMNTs) were fabricated, and characterized by high-resolution transmission electron microscopy, atomic force microscopy, x-ray photoelectron spectroscopy, Raman spectroscopy, Fourier transform infrared spectroscopy and thermogravimetric analysis, revealing the presence of dendrimer capped on the surface of carbon nanotubes. The dMNTs fully conjugated with FITC-labeled antisense c-myc oligonucleotides (asODN), those resultant asODN-dMNTs composites were incubated with human breast cancer cell line MCF-7 cells and MDA-MB-435 cells, and liver cancer cell line HepG2 cells, and confirmed to enter into tumor cells within 15 min by laser confocal microscopy. These composites inhibited the cell growth in time- and dose-dependent means, and down-regulated the expression of the c-myc gene and C-Myc protein. Compared with the composites of CNT-NH2-asODN and dendrimer-asODN, no. 5 generation of dendrimer-modified MNT-asODN composites exhibit maximal transfection efficiencies and inhibition effects on tumor cells. The intracellular gene transport and uptake via dMNTs should be generic for the mammalian cell lines. The dMNTs have potentials in applications such as gene or drug delivery for cancer therapy and molecular imaging.

Transcription factor clusters regulate genes in eukaryotic cells

PubMed Central

Hedlund, Erik G; Friemann, Rosmarie; Hohmann, Stefan

2017-01-01

Transcription is regulated through binding factors to gene promoters to activate or repress expression, however, the mechanisms by which factors find targets remain unclear. Using single-molecule fluorescence microscopy, we determined in vivo stoichiometry and spatiotemporal dynamics of a GFP tagged repressor, Mig1, from a paradigm signaling pathway of Saccharomyces cerevisiae. We find the repressor operates in clusters, which upon extracellular signal detection, translocate from the cytoplasm, bind to nuclear targets and turnover. Simulations of Mig1 configuration within a 3D yeast genome model combined with a promoter-specific, fluorescent translation reporter confirmed clusters are the functional unit of gene regulation. In vitro and structural analysis on reconstituted Mig1 suggests that clusters are stabilized by depletion forces between intrinsically disordered sequences. We observed similar clusters of a co-regulatory activator from a different pathway, supporting a generalized cluster model for transcription factors that reduces promoter search times through intersegment transfer while stabilizing gene expression. PMID:28841133

Quercetin manipulates the expression of genes involved in the reactive oxygen species (ROS) processin chicken heterophils.

PubMed

Nambooppha, Boondarika; Photichai, Kornravee; Wongsawan, Kanreuthai; Chuammitri, Phongsakorn

2018-06-06

Chicken heterophils generate reactive oxygen species (ROS) molecules to defend against invading pathogens. The present study examined effects of quercetin on chicken heterophils. Heterophils were stimulated with PBS, 50 μM quercetin (QH), PMA or Escherichia coli (EC) and the resulting intracellular ROS molecules were determined. Flow cytometry results showed that cells stimulated with QH, PMA and EC had a higher ROS production. Increases in intracellular ROS molecules were identified in all treatment groups by fluorescence microscopy. Determination of the ability of quercetin to manipulate mRNA expression of ROS subunits was assessed using real-time RT-PCR. Quercetin and other stimulants up-regulated the majority of genes involved in ROS production: CYBB (NOX2), NCF1 (p47 phox ), NCF2 (p67 phox ), NOX1 and RAC2. The antioxidant property of QH was explored by measuring mRNA expression of CAT and SOD1. The data indicate increased levels of CAT with all treatments; however, only QH attenuated the expression ofthe SOD1 gene. To further investigate the effects of ROS-driven inflammation or cell death, IL6, CASP8, and MCL1 genes were preferentially tested. The inflammatory gene (IL6) was profoundly down-regulated in the QH- and PMA-treated groups while EC induced a strikingly high IL6 expression level. Investigation of the known apoptotic (CASP8) and anti-apoptotic (MCL1) genes found down-regulation of CASP8 in the QH- and PMA-treated groups which were contradicted to the MCL1 gene. In conclusion, quercetin can enhance ROS production by regulating the expression of genes involved in ROS production as well as in subsequent processes.

Temporal, spatial, and phenotypical changes of PDGFRα expressing fibroblasts during late lung development.

PubMed

Endale, Mehari; Ahlfeld, Shawn; Bao, Erik; Chen, Xiaoting; Green, Jenna; Bess, Zach; Weirauch, Matthew T; Xu, Yan; Perl, Anne Karina

2017-05-15

Many studies have investigated the source and role of epithelial progenitors during lung development; such information is limited for fibroblast populations and their complex role in the developing lung. In this study, we characterized the spatial location, mRNA expression and Immunophenotyping of PDGFRα + fibroblasts during sacculation and alveolarization. Confocal microscopy identified spatial association of PDGFRα expressing fibroblasts with proximal epithelial cells of the branching bronchioles and the dilating acinar tubules at E16.5; with distal terminal saccules at E18.5; and with alveolar epithelial cells at PN7 and PN28. Immunohistochemistry for alpha smooth muscle actin revealed that PDGFRα + fibroblasts contribute to proximal peribronchiolar smooth muscle at E16.5 and to transient distal alveolar myofibroblasts at PN7. Time series RNA-Seq analyses of PDGFRα + fibroblasts identified differentially expressed genes that, based on gene expression similarity were clustered into 7 major gene expression profile patterns. The presence of myofibroblast and smooth muscle precursors at E16.5 and PN7 was reflected by a two-peak gene expression profile on these days and gene ontology enrichment in muscle contraction. Additional molecular and functional differences between peribronchiolar smooth muscle cells at E16.5 and transient intraseptal myofibroblasts at PN7 were suggested by a single peak in gene expression at PN7 with functional enrichment in cell projection and muscle cell differentiation. Immunophenotyping of subsets of PDGFRα + fibroblasts by flow cytometry confirmed the predicted increase in proliferation at E16.5 and PN7, and identified subsets of CD29 + myofibroblasts and CD34 + lipofibroblasts. These data can be further mined to develop novel hypotheses and valuable understanding of the molecular and cellular basis of alveolarization. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Temporal, spatial, and phenotypical changes of PDGFRα expressing fibroblasts during late lung development☆

PubMed Central

Endale, Mehari; Ahlfeld, Shawn; Bao, Erik; Chen, Xiaoting; Green, Jenna; Bess, Zach; Weirauch, Matthew T.; Xu, Yan; Perl, Anne Karina

2017-01-01

Many studies have investigated the source and role of epithelial progenitors during lung development; such information is limited for fibroblast populations and their complex role in the developing lung. In this study, we characterized the spatial location, mRNA expression and Immunophenotyping of PDGFRα+ fibroblasts during sacculation and alveolarization. Confocal microscopy identified spatial association of PDGFRα expressing fibroblasts with proximal epithelial cells of the branching bronchioles and the dilating acinar tubules at E16.5; with distal terminal saccules at E18.5; and with alveolar epithelial cells at PN7 and PN28. Immunohistochemistry for alpha smooth muscle actin revealed that PDGFRα+ fibroblasts contribute to proximal peribronchiolar smooth muscle at E16.5 and to transient distal alveolar myofibroblasts at PN7. Time series RNA-Seq analyses of PDGFRα+ fibroblasts identified differentially expressed genes that, based on gene expression similarity were clustered into 7 major gene expression profile patterns. The presence of myofibroblast and smooth muscle precursors at E16.5 and PN7 was reflected by a two-peak gene expression profile on these days and gene ontology enrichment in muscle contraction. Additional molecular and functional differences between peribronchiolar smooth muscle cells at E16.5 and transient intraseptal myofibroblasts at PN7 were suggested by a single peak in gene expression at PN7 with functional enrichment in cell projection and muscle cell differentiation. Immunophenotyping of subsets of PDGFRα+ fibroblasts by flow cytometry confirmed the predicted increase in proliferation at E16.5 and PN7, and identified subsets of CD29+ myofibroblasts and CD34+ lipofibroblasts. These data can be further mined to develop novel hypotheses and valuable understanding of the molecular and cellular basis of alveolarization. PMID:28408205

Macromolecular Crowding Induces Spatial Correlations That Control Gene Expression Bursting Patterns.

PubMed

Norred, S Elizabeth; Caveney, Patrick M; Chauhan, Gaurav; Collier, Lauren K; Collier, C Patrick; Abel, Steven M; Simpson, Michael L

2018-05-18

Recent superresolution microscopy studies in E. coli demonstrate that the cytoplasm has highly variable local concentrations where macromolecular crowding plays a central role in establishing membrane-less compartmentalization. This spatial inhomogeneity significantly influences molecular transport and association processes central to gene expression. Yet, little is known about how macromolecular crowding influences gene expression bursting-the episodic process where mRNA and proteins are produced in bursts. Here, we simultaneously measured mRNA and protein reporters in cell-free systems, showing that macromolecular crowding decoupled the well-known relationship between fluctuations in the protein population (noise) and mRNA population statistics. Crowded environments led to a 10-fold increase in protein noise even though there were only modest changes in the mRNA population and fluctuations. Instead, cell-like macromolecular crowding created an inhomogeneous spatial distribution of mRNA ("spatial noise") that led to large variability in the protein production burst size. As a result, the mRNA spatial noise created large temporal fluctuations in the protein population. These results highlight the interplay between macromolecular crowding, spatial inhomogeneities, and the resulting dynamics of gene expression, and provide insights into using these organizational principles in both cell-based and cell-free synthetic biology.

Stable chloroplast transformation of immature scutella and inflorescences in wheat (Triticum aestivum L.).

PubMed

Cui, Cuiju; Song, Fei; Tan, Yi; Zhou, Xuan; Zhao, Wen; Ma, Fengyun; Liu, Yunyi; Hussain, Javeed; Wang, Yuesheng; Yang, Guangxiao; He, Guangyuan

2011-04-01

Chloroplast transformation in wheat was achieved by bombardment of scutella from immature embryos and immature inflorescences, respectively. A wheat chloroplast site-specific expression vector, pBAGNRK, was constructed by placing an expression cassette containing neomycin phosphotransferase II (nptII) and green fluorescent protein (gfp) as selection and reporter genes, respectively, in the intergenic spacer between atpB and rbcL of wheat chloroplast genome. Integration of gfp gene in the plastome was identified by polymerase chain reaction (PCR) analysis and Southern blotting using gfp gene as a probe. Expression of GFP protein was examined by western blot. Three positive transformants were obtained and the Southern blot of partial fragment of atpB and rbcL (targeting site) probes verified that one of them was homoplasmic. Stable expression of GFP fluorescence was confirmed by confocal microscopy in the leaf tissues from T(1) progeny seedlings. PCR analysis of gfp gene also confirmed the inheritance of transgene in the T(1) progeny. These results strengthen the feasibility of wheat chloroplast transformation and also give a novel method for the introduction of important agronomic traits in wheat through chloroplast transformation.

Following Pathogen Development and Gene Expression in a Food Ecosystem: the Case of a Staphylococcus aureus Isolate in Cheese

PubMed Central

Aigle, Marina; Fleurot, Renaud; Darrigo, Claire; Hennekinne, Jacques-Antoine; Gruss, Alexandra; Borezée-Durant, Elise; Delacroix-Buchet, Agnès

2014-01-01

Human intoxication or infection due to bacterial food contamination constitutes an economic challenge and a public health problem. Information on the in situ distribution and expression of pathogens responsible for this risk is to date lacking, largely because of technical bottlenecks in detecting signals from minority bacterial populations within a complex microbial and physicochemical ecosystem. We simulated the contamination of a real high-risk cheese with a natural food isolate of Staphylococcus aureus, an enterotoxin-producing pathogen responsible for food poisoning. To overcome the problem of a detection limit in a solid matrix, we chose to work with a fluorescent reporter (superfolder green fluorescent protein) that would allow spatiotemporal monitoring of S. aureus populations and targeted gene expression. The combination of complementary techniques revealed that S. aureus localizes preferentially on the cheese surface during ripening. Immunochemistry and confocal laser scanning microscopy enabled us to visualize, in a single image, dairy bacteria and pathogen populations, virulence gene expression, and the toxin produced. This procedure is readily applicable to other genes of interest, other bacteria, and different types of food matrices. PMID:24928871

NBCe1 expression is required for normal renal ammonia metabolism

PubMed Central

Handlogten, Mary E.; Osis, Gunars; Lee, Hyun-Wook; Romero, Michael F.; Verlander, Jill W.

2015-01-01

The mechanisms regulating proximal tubule ammonia metabolism are incompletely understood. The present study addressed the role of the proximal tubule basolateral electrogenic Na+-coupled bicarbonate cotransporter (NBCe1; Slc4a4) in renal ammonia metabolism. We used mice with heterozygous and homozygous NBCe1 gene deletion and compared these mice with their wild-type littermates. Because homozygous NBCe1 gene deletion causes 100% mortality before day 25, we studied mice at day 8 (±1 day). Both heterozygous and homozygous gene deletion caused a gene dose-related decrease in serum bicarbonate. The ability to lower urinary pH was intact, and even accentuated, with NBCe1 deletion. However, in contrast to the well-known effect of metabolic acidosis to increase urinary ammonia excretion, NBCe1 deletion caused a gene dose-related decrease in ammonia excretion. There was no identifiable change in proximal tubule structure by light microscopy. Examination of proteins involved in renal ammonia metabolism showed decreased expression of phosphate-dependent glutaminase and phosphoenolpyruvate carboxykinase, key enzymes in proximal tubule ammonia generation, and increased expression of glutamine synthetase, which recycles intrarenal ammonia and regenerates glutamine. Expression of key proteins involved in ammonia transport outside of the proximal tubule (rhesus B glycoprotein and rhesus C glycoprotein) was not significantly changed by NBCe1 deletion. We conclude from these findings that NBCe1 expression is necessary for normal proximal tubule ammonia metabolism. PMID:26224717

Revealing New Mouse Epicardial Cell Markers through Transcriptomics

PubMed Central

Bochmann, Lars; Sarathchandra, Padmini; Mori, Federica; Lara-Pezzi, Enrique; Lazzaro, Domenico; Rosenthal, Nadia

2010-01-01

Background The epicardium has key functions during myocardial development, by contributing to the formation of coronary endothelial and smooth muscle cells, cardiac fibroblasts, and potentially cardiomyocytes. The epicardium plays a morphogenetic role by emitting signals to promote and maintain cardiomyocyte proliferation. In a regenerative context, the adult epicardium might comprise a progenitor cell population that can be induced to contribute to cardiac repair. Although some genes involved in epicardial function have been identified, a detailed molecular profile of epicardial gene expression has not been available. Methodology Using laser capture microscopy, we isolated the epicardial layer from the adult murine heart before or after cardiac infarction in wildtype mice and mice expressing a transgenic IGF-1 propeptide (mIGF-1) that enhances cardiac repair, and analyzed the transcription profile using DNA microarrays. Principal Findings Expression of epithelial genes such as basonuclin, dermokine, and glycoprotein M6A are highly enriched in the epicardial layer, which maintains expression of selected embryonic genes involved in epicardial development in mIGF-1 transgenic hearts. After myocardial infarct, a subset of differentially expressed genes are down-regulated in the epicardium representing an epicardium-specific signature that responds to injury. Conclusion This study presents the description of the murine epicardial transcriptome obtained from snap frozen tissues, providing essential information for further analysis of this important cardiac cell layer. PMID:20596535

Genomic Approach to Study Floral Development Genes in Rosa sp.

PubMed Central

Chauvet, Aurélie; Maene, Marion; Pécrix, Yann; Yang, Shu-Hua; Jeauffre, Julien; Thouroude, Tatiana; Boltz, Véronique; Martin-Magniette, Marie-Laure; Janczarski, Stéphane; Legeai, Fabrice; Renou, Jean-Pierre; Vergne, Philippe; Le Bris, Manuel; Foucher, Fabrice; Bendahmane, Mohammed

2011-01-01

Cultivated for centuries, the varieties of rose have been selected based on a number of flower traits. Understanding the genetic and molecular basis that contributes to these traits will impact on future improvements for this economically important ornamental plant. In this study, we used scanning electron microscopy and sections of meristems and flowers to establish a precise morphological calendar from early rose flower development stages to senescing flowers. Global gene expression was investigated from floral meristem initiation up to flower senescence in three rose genotypes exhibiting contrasted floral traits including continuous versus once flowering and simple versus double flower architecture, using a newly developed Affymetrix microarray (Rosa1_Affyarray) tool containing sequences representing 4765 unigenes expressed during flower development. Data analyses permitted the identification of genes associated with floral transition, floral organs initiation up to flower senescence. Quantitative real time PCR analyses validated the mRNA accumulation changes observed in microarray hybridizations for a selection of 24 genes expressed at either high or low levels. Our data describe the early flower development stages in Rosa sp, the production of a rose microarray and demonstrate its usefulness and reliability to study gene expression during extensive development phases, from the vegetative meristem to the senescent flower. PMID:22194838

A part toolbox to tune genetic expression in Bacillus subtilis

PubMed Central

Guiziou, Sarah; Sauveplane, Vincent; Chang, Hung-Ju; Clerté, Caroline; Declerck, Nathalie; Jules, Matthieu; Bonnet, Jerome

2016-01-01

Libraries of well-characterised components regulating gene expression levels are essential to many synthetic biology applications. While widely available for the Gram-negative model bacterium Escherichia coli, such libraries are lacking for the Gram-positive model Bacillus subtilis, a key organism for basic research and biotechnological applications. Here, we engineered a genetic toolbox comprising libraries of promoters, Ribosome Binding Sites (RBS), and protein degradation tags to precisely tune gene expression in B. subtilis. We first designed a modular Expression Operating Unit (EOU) facilitating parts assembly and modifications and providing a standard genetic context for gene circuits implementation. We then selected native, constitutive promoters of B. subtilis and efficient RBS sequences from which we engineered three promoters and three RBS sequence libraries exhibiting ∼14 000-fold dynamic range in gene expression levels. We also designed a collection of SsrA proteolysis tags of variable strength. Finally, by using fluorescence fluctuation methods coupled with two-photon microscopy, we quantified the absolute concentration of GFP in a subset of strains from the library. Our complete promoters and RBS sequences library comprising over 135 constructs enables tuning of GFP concentration over five orders of magnitude, from 0.05 to 700 μM. This toolbox of regulatory components will support many research and engineering applications in B. subtilis. PMID:27402159

Highly Tissue Substructure-Specific Effects of Human Papilloma Virus in Mucosa of HIV-Infected Patients Revealed by Laser-Dissection Microscopy-Assisted Gene Expression Profiling

PubMed Central

Baumgarth, Nicole; Szubin, Richard; Dolganov, Greg M.; Watnik, Mitchell R.; Greenspan, Deborah; Da Costa, Maria; Palefsky, Joel M.; Jordan, Richard; Roederer, Mario; Greenspan, John S.

2004-01-01

Human papilloma virus (HPV) causes focal infections of epithelial layers in skin and mucosa. HIV-infected patients on highly active antiretroviral therapy (HAART) appear to be at increased risk of developing HPV-induced oral warts. To identify the mechanisms that allow long-term infection of oral epithelial cells in these patients, we used a combination of laser-dissection microscopy (LDM) and highly sensitive and quantitative, non-biased, two-step multiplex real-time RT-PCR to study pathogen-induced alterations of specific tissue subcompartments. Expression of 166 genes was compared in three distinct epithelial and subepithelial compartments isolated from biopsies of normal mucosa from HIV-infected and non-infected patients and of HPV32-induced oral warts from HIV-infected patients. In contrast to the underlying HIV infection and/or HAART, which did not significantly elaborate tissue substructure-specific effects, changes in oral warts were strongly tissue substructure-specific. HPV 32 seems to establish infection by selectively enhancing epithelial cell growth and differentiation in the stratum spinosum and to evade the immune system by actively suppressing inflammatory responses in adjacent underlying tissues. With this highly sensitive and quantitative method tissue-specific expression of hundreds of genes can be studied simultaneously in a few cells. Because of its large dynamic measurement range it could also become a method of choice to confirm and better quantify results obtained by microarray analysis. PMID:15331396

An evaluation of chondrocyte morphology and gene expression on superhydrophilic vertically-aligned multi-walled carbon nanotube films.

PubMed

Antonioli, Eliane; Lobo, Anderson O; Ferretti, Mario; Cohen, Moisés; Marciano, Fernanda R; Corat, Evaldo J; Trava-Airoldi, Vladimir J

2013-03-01

Cartilage serves as a low-friction and wear-resistant articulating surface in diarthrodial joints and is also important during early stages of bone remodeling. Recently, regenerative cartilage research has focused on combinations of cells paired with scaffolds. Superhydrophilic vertically aligned carbon nanotubes (VACNTs) are of particular interest in regenerative medicine. The aim of this study is to evaluate cell expansion of human articular chondrocytes on superhydrophilic VACNTs, as well as their morphology and gene expression. VACNT films were produced using a microwave plasma chamber on Ti substrates and submitted to an O2 plasma treatment to make them superhydrophilic. Human chondrocytes were cultivated on superhydrophilic VACNTs up to five days. Quantitative RT-PCR was performed to measure type I and type II Collagen, Sox9, and Aggrecan mRNA expression levels. The morphology was analyzed by scanning electron microscopy (SEM) and confocal microscopy. SEM images demonstrated that superhydrophilic VACNTs permit cell growth and adhesion of human chondrocytes. The chondrocytes had an elongated morphology with some prolongations. Chondrocytes cultivated on superhydrophilic VACNTs maintain the level expression of Aggrecan, Sox9, and Collagen II determined by qPCR. This study was the first to indicate that superhydrophilic VACNTs may be used as an efficient scaffold for cartilage or bone repair. Copyright © 2012 Elsevier B.V. All rights reserved.

Artemisia princeps Inhibits Biofilm Formation and Virulence-Factor Expression of Antibiotic-Resistant Bacteria

PubMed Central

Kang, Sun-Young; Kim, Kang-Ju

2015-01-01

In this study, we used ethanol extract of A. princeps and investigated its antibacterial effects against MRSA. Ethanol extract of A. princeps significantly inhibited MRSA growth and organic acid production during glucose metabolism at concentrations greater than 1 mg/mL (P < 0.05). MRSA biofilm formation was observed using scanning electron microscopy (SEM) and safranin staining. A. princeps extract was found to inhibit MRSA biofilm formation at concentrations higher than 2 mg/mL significantly (P < 0.05). Bactericidal effects of the A. princeps were observed using confocal laser microscopy, which showed that A. princeps was bactericidal in a dose-dependent manner. Using real-time PCR, expression of mecA, an antibiotic-resistance gene of MRSA, was observed, along with that of sea, agrA, and sarA. A. princeps significantly inhibited mecA, sea, agrA, and sarA, mRNA expression at the concentrations greater than 1 mg/mL (P < 0.05). The phytochemical analysis of A. princeps showed a relatively high content of organic acids and glycosides. The results of this study suggest that the ethanol extract of A. princeps may inhibit proliferation, acid production, biofilm formation, and virulence gene expressions of MRSA, which may be related to organic acids and glycosides, the major components in the extract. PMID:26247012

Artemisia princeps Inhibits Biofilm Formation and Virulence-Factor Expression of Antibiotic-Resistant Bacteria.

PubMed

Choi, Na-Young; Kang, Sun-Young; Kim, Kang-Ju

2015-01-01

In this study, we used ethanol extract of A. princeps and investigated its antibacterial effects against MRSA. Ethanol extract of A. princeps significantly inhibited MRSA growth and organic acid production during glucose metabolism at concentrations greater than 1 mg/mL (P < 0.05). MRSA biofilm formation was observed using scanning electron microscopy (SEM) and safranin staining. A. princeps extract was found to inhibit MRSA biofilm formation at concentrations higher than 2 mg/mL significantly (P < 0.05). Bactericidal effects of the A. princeps were observed using confocal laser microscopy, which showed that A. princeps was bactericidal in a dose-dependent manner. Using real-time PCR, expression of mecA, an antibiotic-resistance gene of MRSA, was observed, along with that of sea, agrA, and sarA. A. princeps significantly inhibited mecA, sea, agrA, and sarA, mRNA expression at the concentrations greater than 1 mg/mL (P < 0.05). The phytochemical analysis of A. princeps showed a relatively high content of organic acids and glycosides. The results of this study suggest that the ethanol extract of A. princeps may inhibit proliferation, acid production, biofilm formation, and virulence gene expressions of MRSA, which may be related to organic acids and glycosides, the major components in the extract.

Liver Cell-Derived Microparticles Activate Hedgehog Signaling and Alter Gene Expression in Hepatic Endothelial Cells

PubMed Central

Witek, Rafal P.; Yang, Liu; Liu, Renshui; Jung, Youngmi; Omenetti, Alessia; Syn, Wing-Kin; Choi, Steve S.; Cheong, Yeiwon; Fearing, Caitlin M.; Agboola, Kolade M.; Chen, Wei; Diehl, Anna Mae

2013-01-01

Background & Aims Angiogenesis contributes to vascular remodeling during cirrhosis. In cirrhotic livers, cholangiocytes and myofibroblastic hepatic stellate cells (MF-HSC) produce Hedgehog (Hh) ligands. During embryogenesis Hh ligands are released from ligand-producing cells in microparticles and activate Hh signaling in endothelial cells. We studied whether adult liver cell-derived microparticles contain Hh ligands that alter hepatic sinusoidal endothelial cells (SEC). Methods MF-HSCs and cholangiocytes were exposed to platelet-derived growth factor (PDGF) to induce Hh ligands; microparticles were isolated from medium, analyzed by transmission electron microscopy (TEM) and immunoblots, and applied to Hh-reporter containing cells. Microparticles were also obtained from serum and bile of rats after bile duct ligation (BDL) or sham surgery and applied to normal primary liver SEC with or without cyclopamine, a Hh signaling inhibitor. Effects on SEC gene expression were evaluated by QRT-PCR and immunoblotting. Finally, Hh target gene expression and SEC activation markers were compared in primary SEC and in liver sections from healthy and BDL rats. Results PDGF-treated MF-HSC and cholangiocytes released exosome-enriched microparticles containing biologically active Hh ligands. BDL also increased release of Hh-containing exosome-enriched microparticles into plasma and bile. TEM and immunoblots revealed similarities among microparticles from all sources; all microparticles induced similar Hh-dependent changes in SEC gene expression. SEC from healthy livers did not express Hh target genes or activation markers, but both were up-regulated in SEC after BDL. Conclusions Hh-containing exosome-enriched microparticles released from liver cells alter hepatic SEC gene expression, suggesting a novel mechanism for cirrhotic vasculopathy. PMID:19013163

Cell Population Kinetics of Collagen Scaffolds in Ex Vivo Oral Wound Repair

PubMed Central

Agis, Hermann; Collins, Amy; Taut, Andrei D.; Jin, Qiming; Kruger, Laura; Görlach, Christoph; Giannobile, William V.

2014-01-01

Biodegradable collagen scaffolds are used clinically for oral soft tissue augmentation to support wound healing. This study sought to provide a novel ex vivo model for analyzing healing kinetics and gene expression of primary human gingival fibroblasts (hGF) within collagen scaffolds. Sponge type and gel type scaffolds with and without platelet-derived growth factor-BB (PDGF) were assessed in an hGF containing matrix. Morphology was evaluated with scanning electron microscopy, and hGF metabolic activity using MTT. We quantitated the population kinetics within the scaffolds based on cell density and distance from the scaffold border of DiI-labled hGFs over a two-week observation period. Gene expression was evaluated with gene array and qPCR. The sponge type scaffolds showed a porous morphology. Absolute cell number and distance was higher in sponge type scaffolds when compared to gel type scaffolds, in particular during the first week of observation. PDGF incorporated scaffolds increased cell numbers, distance, and formazan formation in the MTT assay. Gene expression dynamics revealed the induction of key genes associated with the generation of oral tissue. DKK1, CYR61, CTGF, TGFBR1 levels were increased and integrin ITGA2 levels were decreased in the sponge type scaffolds compared to the gel type scaffold. The results suggest that this novel model of oral wound healing provides insights into population kinetics and gene expression dynamics of biodegradable scaffolds. PMID:25397671

The citrus flavonone hesperetin attenuates the nuclear translocation of aryl hydrocarbon receptor.

PubMed

Tan, Yan Qin; Chiu-Leung, Leo Clement; Lin, Shu-Mei; Leung, Lai K

2018-08-01

The environmental polycyclic aromatic hydrocarbons (PAH) and dioxins are carcinogens and their adverse effects have been largely attributed to the activation of AhR. Hesperetin is a flavonone found abundantly in citrus fruits and has been shown to be a biologically active agent. In the present study, the effect of hesperetin on the nuclear translocation of AhR and the downstream gene expression was investigated in MCF-7 cells. Confocal microscopy indicated that 7, 12-dimethylbenz[α]anthracene (DMBA) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) -induced nuclear translocation of AhR was deterred by hesperetin treatment. The reduced nuclear translocation could also be observed in Western analysis. Reporter-gene assay further illustrated that the induced XRE transactivation was weakened by the treatment of hesperetin. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay demonstrated that the gene expressions of CYP1A1, 1A2, and 1B1 followed the same pattern of AhR translocation. These results suggested that hesperetin counteracted AhR transactivation and suppressed the downstream gene expression. Copyright © 2018 Elsevier Inc. All rights reserved.

Changes in the Salmonella enterica Enteritidis phenotypes in presence of acyl homoserine lactone quorum sensing signals.

PubMed

Campos-Galvão, Maria Emilene Martino; Ribon, Andrea Oliveira Barros; Araújo, Elza Fernandes; Vanetti, Maria Cristina Dantas

2016-05-01

Quorum sensing is used by bacteria to coordinate gene expression in response to population density and involves the production, detection and response to extracellular signaling molecules known as autoinducers (AIs). Salmonella does not synthesize the AI-1, acyl homoserine lactone (AHL) common to gram-negative bacteria; however, it has a receptor for AI-1, the SdiA protein. The effect of SdiA in modulating phenotypes of Salmonella has not been elucidated. In this report, we provide evidence that the AIs-1 affect Salmonella enterica serovar Enteritidis behavior by enhancing the biofilm formation and expression of virulence genes under anaerobic conditions. Biofilm formation by Salmonella was detected by the crystal violet method and by scanning electron microscopy. The presence of AHLs, particularly C12-HSL, increased biofilm formation and promoted expression of biofilm formation genes (lpfA, fimF, fliF, glgC) and virulence genes (hilA, invA, invF). Our results demonstrated that AHLs produced by other organisms played an important role in virulence phenotypes of Salmonella Enteritidis. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization and evaluation of apoptotic potential of double gene construct pVIVO.VP3.NS1.

PubMed

Saxena, Shikha; Desai, G S; Kumar, G Ravi; Sahoo, A P; Santra, Lakshman; Singh, Lakshya Veer

2015-05-01

Viral gene oncotherapy, targeted killing of cancer cells by viral genes, is an emerging non-infectious therapeutic cancer treatment modality. Chemo and radiotherapy in cancer treatment is limited due to their genotoxic side effects on healthy cells and need of functional p53, which is mutated in most of the cancers. VP3 (apoptin) of chicken infectious anaemia (CIA) and NS1 (Non structural protein 1) of Canine Parvovirus-2 (CPV-2) have been proven to have oncolytic potential in our laboratory. To evaluate oncolytic potential of VP3 and NS1 together these genes needed to be cloned in a bicistronic vector. In this study, both these genes were cloned and characterized for expression of their gene products and its apoptotic potential. The expression of VP3 and NS1 was studied by confocal microscopy and flowcytometry. Expression of VP3 and NS1 in pVIVO.VP3.NS1 transfected HeLa cells in comparison to mock transfected cells indicated that the double gene construct expresses both the products. This was further confirmed by flowcytometry where there was increase in cells expressing VP3 and NS1 in pVIVO.VP3.NS1 transfected group in comparison with the mock control group. The apoptotic inducing potential of this characterized pVIVO.VP3.NS1 was evaluated in human cervical cancer cell line (HeLa) by DNA fragmentation assay, TUNEL assay and Hoechst staning. This double construct was observed to induce apoptosis in HeLa cells.

A cell-penetrating peptide analogue, P7, exerts antimicrobial activity against Escherichia coli ATCC25922 via penetrating cell membrane and targeting intracellular DNA.

PubMed

Li, Lirong; Shi, Yonghui; Cheng, Xiangrong; Xia, Shufang; Cheserek, Maureen Jepkorir; Le, Guowei

2015-01-01

The antibacterial activities and mechanism of a new P7 were investigated in this study. P7 showed antimicrobial activities against five harmful microorganisms which contaminate and spoil food (MIC=4-32 μM). Flow cytometry and scanning electron microscopy analyses demonstrated that P7 induced pore-formation on the cell surface and led to morphological changes but did not lyse cell. Confocal fluorescence microscopic observations and flow cytometry analysis expressed that P7 could penetrate the Escherichia coli cell membrane and accumulate in the cytoplasm. Moreover, P7 possessed a strong DNA binding affinity. Further cell cycle analysis and change in gene expression analysis suggested that P7 induced a decreased expression in the genes involved in DNA replication. Up-regulated expression genes encoding DNA damage repair. This study suggests that P7 could be applied as a candidate for the development of new food preservatives as it exerts its antibacterial activities by penetrating cell membranes and targets intracellular DNA. Copyright © 2014 Elsevier Ltd. All rights reserved.

Confocal quantification of cis-regulatory reporter gene expression in living sea urchin.

PubMed

Damle, Sagar; Hanser, Bridget; Davidson, Eric H; Fraser, Scott E

2006-11-15

Quantification of GFP reporter gene expression at single cell level in living sea urchin embryos can now be accomplished by a new method of confocal laser scanning microscopy (CLSM). Eggs injected with a tissue-specific GFP reporter DNA construct were grown to gastrula stage and their fluorescence recorded as a series of contiguous Z-section slices that spanned the entire embryo. To measure the depth-dependent signal decay seen in the successive slices of an image stack, the eggs were coinjected with a freely diffusible internal fluorescent standard, rhodamine dextran. The measured rhodamine fluorescence was used to generate a computational correction for the depth-dependent loss of GFP fluorescence per slice. The intensity of GFP fluorescence was converted to the number of GFP molecules using a conversion constant derived from CLSM imaging of eggs injected with a measured quantity of GFP protein. The outcome is a validated method for accurately counting GFP molecules in given cells in reporter gene transfer experiments, as we demonstrate by use of an expression construct expressed exclusively in skeletogenic cells.

Upregulation of S1P1 and Rac1 receptors in the pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia.

PubMed

Zimmer, Julia; Takahashi, Toshiaki; Duess, Johannes W; Hofmann, Alejandro D; Puri, Prem

2016-02-01

Sphingolipids play a crucial role in pulmonary development. The sphingosine kinase 1 (SphK1) modulates the synthesis of sphingolipid sphingosine-1-phosphate (S1P). S1P regulates cell proliferation and angiogenesis via different receptors, S1P1, S1P2 and S1P3, which all influence the expression of Ras-related C3 botulinum toxin substrate 1 (Rac1). We designed this study to test the hypothesis that the S1P/Rac1 pathway is altered in the nitrofen-induced CDH model. Pregnant rats received nitrofen or vehicle on D9. On D21, fetuses were killed and divided into nitrofen and control group (n = 12). QRT-PCR, western blotting and confocal-immunofluorescence microscopy were performed to reveal pulmonary gene and protein expression levels of SphK1, S1P1, S1P2, S1P3 and Rac1. Pulmonary gene expression of S1P1 and Rac1 was significantly increased in the CDH group compared to controls, whereas S1P2 and S1P3 expression was decreased. These results were confirmed by western blotting and confocal microscopy. SphK1 expression was not found to be altered. The increased expression of S1P1 and Rac1 in the pulmonary vasculature of nitrofen-induced CDH lungs suggests that S1P1 and Rac1 are important mediators of PH in this model.

Enhanced green fluorescent protein (egfp) gene expression in Tetraselmis subcordiformis chloroplast with endogenous regulators.

PubMed

Cui, Yulin; Zhao, Jialin; Hou, Shichang; Qin, Song

2016-05-01

On the basis of fundamental genetic transformation technologies, the goal of this study was to optimize Tetraselmis subcordiformis chloroplast transformation through the use of endogenous regulators. The genes rrn16S, rbcL, psbA, and psbC are commonly highly expressed in chloroplasts, and the regulators of these genes are often used in chloroplast transformation. For lack of a known chloroplast genome sequence, the genome-walking method was used here to obtain full sequences of T. subcordiformis endogenous regulators. The resulting regulators, including three promoters, two terminators, and a ribosome combination sequence, were inserted into the previously constructed plasmid pPSC-R, with the egfp gene included as a reporter gene, and five chloroplast expression vectors prepared. These vectors were successfully transformed into T. subcordiformis by particle bombardment and the efficiency of each vector tested by assessing EGFP fluorescence via microscopy. The results showed that these vectors exhibited higher efficiency than the former vector pPSC-G carrying exogenous regulators, and the vector pRFA with Prrn, psbA-5'RE, and TpsbA showed the highest efficiency. This research provides a set of effective endogenous regulators for T. subcordiformis and will facilitate future fundamental studies of this alga.

RNA-seq transcriptional profiling of Herbaspirillum seropedicae colonizing wheat (Triticum aestivum) roots.

PubMed

Pankievicz, V C S; Camilios-Neto, D; Bonato, P; Balsanelli, E; Tadra-Sfeir, M Z; Faoro, H; Chubatsu, L S; Donatti, L; Wajnberg, G; Passetti, F; Monteiro, R A; Pedrosa, F O; Souza, E M

2016-04-01

Herbaspirillum seropedicae is a diazotrophic and endophytic bacterium that associates with economically important grasses promoting plant growth and increasing productivity. To identify genes related to bacterial ability to colonize plants, wheat seedlings growing hydroponically in Hoagland's medium were inoculated with H. seropedicae and incubated for 3 days. Total mRNA from the bacteria present in the root surface and in the plant medium were purified, depleted from rRNA and used for RNA-seq profiling. RT-qPCR analyses were conducted to confirm regulation of selected genes. Comparison of RNA profile of root attached and planktonic bacteria revealed extensive metabolic adaptations to the epiphytic life style. These adaptations include expression of specific adhesins and cell wall re-modeling to attach to the root. Additionally, the metabolism was adapted to the microxic environment and nitrogen-fixation genes were expressed. Polyhydroxybutyrate (PHB) synthesis was activated, and PHB granules were stored as observed by microscopy. Genes related to plant growth promotion, such as auxin production were expressed. Many ABC transporter genes were regulated in the bacteria attached to the roots. The results provide new insights into the adaptation of H. seropedicae to the interaction with the plant.

Targeted Gene Editing of Glia Maturation Factor in Microglia: a Novel Alzheimer's Disease Therapeutic Target.

PubMed

Raikwar, Sudhanshu P; Thangavel, Ramasamy; Dubova, Iuliia; Selvakumar, Govindhasamy Pushpavathi; Ahmed, Mohammad Ejaz; Kempuraj, Duraisamy; Zaheer, Smita A; Iyer, Shankar S; Zaheer, Asgar

2018-04-27

Alzheimer's disease (AD) is a devastating, progressive neurodegenerative disorder that leads to severe cognitive impairment in elderly patients. Chronic neuroinflammation plays an important role in the AD pathogenesis. Glia maturation factor (GMF), a proinflammatory molecule discovered in our laboratory, is significantly upregulated in various regions of AD brains. We have previously reported that GMF is predominantly expressed in the reactive glial cells surrounding the amyloid plaques (APs) in the mouse and human AD brain. Microglia are the major source of proinflammatory cytokines and chemokines including GMF. Recently clustered regularly interspaced short palindromic repeats (CRISPR) based genome editing has been recognized to study the functions of genes that are implicated in various diseases. Here, we investigated if CRISPR-Cas9-mediated GMF gene editing leads to inhibition of GMF expression and suppression of microglial activation. Confocal microscopy of murine BV2 microglial cell line transduced with an adeno-associated virus (AAV) coexpressing Staphylococcus aureus (Sa) Cas9 and a GMF-specific guide RNA (GMF-sgRNA) revealed few cells expressing SaCas9 while lacking GMF expression, thereby confirming successful GMF gene editing. To further improve GMF gene editing efficiency, we developed lentiviral vectors (LVs) expressing either Streptococcus pyogenes (Sp) Cas9 or GMF-sgRNAs. BV2 cells cotransduced with LVs expressing SpCas9 and GMF-sgRNAs revealed reduced GMF expression and the presence of indels in the exons 2 and 3 of the GMF coding sequence. Lipopolysaccharide (LPS) treatment of GMF-edited cells led to reduced microglial activation as shown by reduced p38 MAPK phosphorylation. We believe that targeted in vivo GMF gene editing has a significant potential for developing a unique and novel AD therapy.

Comparative analysis of surface wax in mature fruits between Satsuma mandarin (Citrus unshiu) and 'Newhall' navel orange (Citrus sinensis) from the perspective of crystal morphology, chemical composition and key gene expression.

PubMed

Wang, Jinqiu; Hao, Haohao; Liu, Runsheng; Ma, Qiaoli; Xu, Juan; Chen, Feng; Cheng, Yunjiang; Deng, Xiuxin

2014-06-15

Surface wax of mature Satsuma mandarin (Citrus unshiu) and 'Newhall' navel orange (Citrus sinensis) was analysed by crystal morphology, chemical composition, and gene expression levels. The epicuticular and total waxes of both citrus cultivars were mostly composed of aldehydes, alkanes, fatty acids and primary alcohols. The epicuticular wax accounted for 80% of the total wax in the Newhall fruits and was higher than that in the Satsuma fruits. Scanning electron microscopy showed that larger and more wax platelets were deposited on the surface of Newhall fruits than on the Satsuma fruits. Moreover, the expression levels of genes involved in the wax formation were consistent with the biochemical and crystal morphological analyses. These diversities of fruit wax between the two cultivars may contribute to the differences of fruit postharvest storage properties, which can provide important information for the production of synthetic wax for citrus fruits. Copyright © 2013 Elsevier Ltd. All rights reserved.

ClearSee: a rapid optical clearing reagent for whole-plant fluorescence imaging

PubMed Central

Kurihara, Daisuke; Mizuta, Yoko; Sato, Yoshikatsu; Higashiyama, Tetsuya

2015-01-01

Imaging techniques for visualizing and analyzing precise morphology and gene expression patterns are essential for understanding biological processes during development in all organisms. With the aid of chemical screening, we developed a clearing method using chemical solutions, termed ClearSee, for deep imaging of morphology and gene expression in plant tissues. ClearSee rapidly diminishes chlorophyll autofluorescence while maintaining fluorescent protein stability. By adjusting the refractive index mismatch, whole-organ and whole-plant imaging can be performed by both confocal and two-photon excitation microscopy in ClearSee-treated samples. Moreover, ClearSee is applicable to multicolor imaging of fluorescent proteins to allow structural analysis of multiple gene expression. Given that ClearSee is compatible with staining by chemical dyes, the technique is useful for deep imaging in conjunction with genetic markers and for plant species not amenable to transgenic approaches. This method is useful for whole imaging for intact morphology and will help to accelerate the discovery of new phenomena in plant biological research. PMID:26493404

Effect of sporophytic PIRL9 genotype on post-meiotic expression of the Arabidopsis pirl1;pirl9 mutant pollen phenotype.

PubMed

Forsthoefel, Nancy R; Vernon, Daniel M

2011-02-01

Plant intracellular ras-group-related leucine-rich repeat proteins (PIRLs) are a novel class of plant leucine-rich repeat (LRR) proteins structurally related to animal ras-group LRRs involved in cell signaling and gene regulation. Gene knockout analysis has shown that two members of the Arabidopsis thaliana PIRL gene family, PIRL1 and PIRL9, are redundant and essential for pollen development and viability: pirl1;pirl9 microspores produced by pirl1/PIRL1;pirl9 plants consistently abort just before pollen mitosis I. qrt1 tetrad analysis demonstrated that the genes become essential after meiosis, during anther stage 10. In this study, we characterized the phenotype of pirl1;pirl9 pollen produced by plants heterozygous for pirl9 (pirl1;pirl9/PIRL9). Alexander's staining, scanning electron microscopy, and fluorescence microscopy indicated that pirl1;pirl9 double mutants produced by pirl9 heterozygotes have a less severe phenotype and more variable morphology than pirl1;pirl9 pollen from pirl1/PIRL1;pirl9 plants. Mutant pollen underwent developmental arrest with variable timing, often progressing beyond pollen mitosis I and arresting at the binucleate stage. Thus, although the pirl1 and pirl9 mutations act post-meiosis, the timing and expressivity of the pirl1;pirl9 pollen phenotype depends on the pirl9 genotype of the parent plant. These results suggest a continued requirement for PIRL1 and PIRL9 beyond the initiation of pollen mitosis. Furthermore, they reveal a modest but novel sporophytic effect in which parent plant genotype influences a mutant phenotype expressed in the haploid generation.

Antibacterial activity and mechanism of action of ε-poly-L-lysine.

PubMed

Ye, Ruosong; Xu, Hengyi; Wan, Cuixiang; Peng, Shanshan; Wang, Lijun; Xu, Hong; Aguilar, Zoraida P; Xiong, Yonghua; Zeng, Zheling; Wei, Hua

2013-09-13

ε-Poly-L-lysine (ε-PL)(2) is widely used as an antibacterial agent because of its broad antimicrobial spectrum. However, the mechanism of ε-PL against pathogens at the molecular level has not been elucidated. This study investigated the antibacterial activity and mechanism of ε-PL against Escherichia coli O157:H7 CMCC44828. Propidium monoazide-PCR test results indicated that the threshold condition of ε-PL for complete membrane lysis of E. coli O157:H7 was 10 μg/mL (90% mortality for 5 μg/mL). Further verification of the destructive effect of ε-PL on cell structure was performed by atomic force microscopy and transmission electron microscopy. Results showed a positive correlation between reactive oxygen species (ROS)(3) levels and ε-PL concentration in E. coli O157:H7 cells. Moreover, the mortality of E. coli O157:H7 was reduced when antioxidant N-acetylcysteine was added. Results from real-time quantitative PCR (RT-qPCR)(4) indicated that the expression levels of oxidative stress genes sodA and oxyR were up-regulated 4- and 16-fold, respectively, whereas virulence genes eaeA and espA were down-regulated after ε-PL treatment. Expression of DNA damage response (SOS response)(5) regulon genes recA and lexA were also affected by ε-PL. In conclusion, the antibacterial mechanism of ε-PL against E. coli O157:H7 may be attributed to disturbance on membrane integrity, oxidative stress by ROS, and effects on various gene expressions, such as regulation of oxidative stress, SOS response, and changes in virulence. Copyright © 2013. Published by Elsevier Inc.

Gene gun bombardment-mediated expression and translocation of EGFP-tagged GLUT4 in skeletal muscle fibres in vivo.

PubMed

Lauritzen, Hans P M M; Reynet, Christine; Schjerling, Peter; Ralston, Evelyn; Thomas, Stephen; Galbo, Henrik; Ploug, Thorkil

2002-09-01

Cellular protein trafficking has been studied to date only in vitro or with techniques that are invasive and have a low time resolution. To establish a gentle method for analysis of glucose transporter-4 (GLUT4) trafficking in vivo in fully differentiated rat skeletal muscle fibres we combined the enhanced green fluorescent protein (EGFP) labelling technique with physical transfection methods in vivo: intramuscular plasmid injection or gene gun bombardment. During optimisation experiments with plasmid coding for the EGFP reporter alone EGFP-positive muscle fibres were counted after collagenase treatment of in vivo transfected flexor digitorum brevis (FDB) muscles. In contrast to gene gun bombardment, intramuscular injection produced EGFP expression in only a few fibres. Regardless of the transfection technique, EGFP expression was higher in muscles from 2-week-old rats than in those from 6-week-old rats and peaked around 1 week after transfection. The gene gun was used subsequently with a plasmid coding for EGFP linked to the C-terminus of GLUT4 (GLUT4-EGFP). Rats were anaesthetised 5 days after transfection and insulin given i.v. with or without accompanying electrical hindleg muscle stimulation. After stimulation, the hindlegs were fixed by perfusion. GLUT4-EGFP-positive FDB fibres were isolated and analysed by confocal microscopy. The intracellular distribution of GLUT4-EGFP under basal conditions as well as after translocation to the plasma membrane in response to insulin, contractions, or both, was in accordance with previous studies of endogenous GLUT4. Finally, GLUT4-EGFP trafficking in quadriceps muscle in vivo was studied using time-lapse microscopy analysis in anaesthetised mice and the first detailed time-lapse recordings of GLUT4-EGFP translocation in fully differentiated skeletal muscle in vivo were obtained.

Purification of recombinant budgerigar fledgling disease virus VP1 capsid protein and its ability for in vitro capsid assembly

NASA Technical Reports Server (NTRS)

Rodgers, R. E.; Chang, D.; Cai, X.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

1994-01-01

A recombinant system for the major capsid VP1 protein of budgerigar fledgling disease virus has been established. The VP1 gene was inserted into a truncated form of the pFlag-1 vector and expressed in Escherichia coli. The budgerigar fledgling disease virus VP1 protein was purified to near homogeneity by immunoaffinity chromatography. Fractions containing highly purified VP1 were pooled and found to constitute 3.3% of the original E. coli-expressed VP1 protein. Electron microscopy revealed that the VP1 protein was isolated as pentameric capsomeres. Electron microscopy also revealed that capsid-like particles were formed in vitro from purified VP1 capsomeres with the addition of Ca2+ ions and the removal of chelating and reducing agents.

Mutations in spalt cause a severe but reversible neurodegenerative phenotype in the embryonic central nervous system of Drosophila melanogaster.

PubMed

Cantera, Rafael; Lüer, Karin; Rusten, Tor Erik; Barrio, Rosa; Kafatos, Fotis C; Technau, Gerhard M

2002-12-01

The gene spalt is expressed in the embryonic central nervous system of Drosophila melanogaster but its function in this tissue is still unknown. To investigate this question, we used a combination of techniques to analyse spalt mutant embryos. Electron microscopy showed that in the absence of spalt, the central nervous system cells are separated by enlarged extracellular spaces populated by membranous material at 60% of embryonic development. Surprisingly, the central nervous system from slightly older embryos (80% of development) exhibited almost wild-type morphology. An extensive survey by laser confocal microscopy revealed that the spalt mutant central nervous system has abnormal levels of particular cell adhesion and cytoskeletal proteins. Time-lapse analysis of neuronal differentiation in vitro, lineage analysis and transplantation experiments confirmed that the mutation causes cytoskeletal and adhesion defects. The data indicate that in the central nervous system, spalt operates within a regulatory pathway which influences the expression of the beta-catenin Armadillo, its ligand N-Cadherin, Notch, and the cell adhesion molecules Neuroglian, Fasciclin 2 and Fasciclin 3. Effects on the expression of these genes are persistent but many morphological aspects of the phenotype are transient, leading to the concept of sequential redundancy for stable organisation of the central nervous system.

nip, a Symbiotic Medicago truncatula Mutant That Forms Root Nodules with Aberrant Infection Threads and Plant Defense-Like Response1

PubMed Central

Veereshlingam, Harita; Haynes, Janine G.; Penmetsa, R. Varma; Cook, Douglas R.; Sherrier, D. Janine; Dickstein, Rebecca

2004-01-01

To investigate the legume-Rhizobium symbiosis, we isolated and studied a novel symbiotic mutant of the model legume Medicago truncatula, designated nip (numerous infections and polyphenolics). When grown on nitrogen-free media in the presence of the compatible bacterium Sinorhizobium meliloti, the nip mutant showed nitrogen deficiency symptoms. The mutant failed to form pink nitrogen-fixing nodules that occur in the wild-type symbiosis, but instead developed small bump-like nodules on its roots that were blocked at an early stage of development. Examination of the nip nodules by light microscopy after staining with X-Gal for S. meliloti expressing a constitutive GUS gene, by confocal microscopy following staining with SYTO-13, and by electron microscopy revealed that nip initiated symbiotic interactions and formed nodule primordia and infection threads. The infection threads in nip proliferated abnormally and very rarely deposited rhizobia into plant host cells; rhizobia failed to differentiate further in these cases. nip nodules contained autofluorescent cells and accumulated a brown pigment. Histochemical staining of nip nodules revealed this pigment to be polyphenolic accumulation. RNA blot analyses demonstrated that nip nodules expressed only a subset of genes associated with nodule organogenesis, as well as elevated expression of a host defense-associated phenylalanine ammonia lyase gene. nip plants were observed to have abnormal lateral roots. nip plant root growth and nodulation responded normally to ethylene inhibitors and precursors. Allelism tests showed that nip complements 14 other M. truncatula nodulation mutants but not latd, a mutant with a more severe nodulation phenotype as well as primary and lateral root defects. Thus, the nip mutant defines a new locus, NIP, required for appropriate infection thread development during invasion of the nascent nodule by rhizobia, normal lateral root elongation, and normal regulation of host defense-like responses during symbiotic interactions. PMID:15516506

Human Antiviral Protein IFIX Suppresses Viral Gene Expression during Herpes Simplex Virus 1 (HSV-1) Infection and Is Counteracted by Virus-induced Proteasomal Degradation.

PubMed

Crow, Marni S; Cristea, Ileana M

2017-04-01

The interferon-inducible protein X (IFIX), a member of the PYHIN family, was recently recognized as an antiviral factor against infection with herpes simplex virus 1 (HSV-1). IFIX binds viral DNA upon infection and promotes expression of antiviral cytokines. How IFIX exerts its host defense functions and whether it is inhibited by the virus remain unknown. Here, we integrated live cell microscopy, proteomics, IFIX domain characterization, and molecular virology to investigate IFIX regulation and antiviral functions during HSV-1 infection. We find that IFIX has a dynamic localization during infection that changes from diffuse nuclear and nucleoli distribution in uninfected cells to discrete nuclear puncta early in infection. This is rapidly followed by a reduction in IFIX protein levels. Indeed, using immunoaffinity purification and mass spectrometry, we define IFIX interactions during HSV-1 infection, finding an association with a proteasome subunit and proteins involved in ubiquitin-proteasome processes. Using synchronized HSV-1 infection, microscopy, and proteasome-inhibition experiments, we demonstrate that IFIX co-localizes with nuclear proteasome puncta shortly after 3 h of infection and that its pyrin domain is rapidly degraded in a proteasome-dependent manner. We further demonstrate that, in contrast to several other host defense factors, IFIX degradation is not dependent on the E3 ubiquitin ligase activity of the viral protein ICP0. However, we show IFIX degradation requires immediate-early viral gene expression, suggesting a viral host suppression mechanism. The IFIX interactome also demonstrated its association with transcriptional regulatory proteins, including the 5FMC complex. We validate this interaction using microscopy and reciprocal isolations and determine it is mediated by the IFIX HIN domain. Finally, we show IFIX suppresses immediate-early and early viral gene expression during infection. Altogether, our study demonstrates that IFIX antiviral functions work in part via viral transcriptional suppression and that HSV-1 has acquired mechanisms to block its functions via proteasome-dependent degradation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Role of host microenvironment in angiogenesis and microvascular functions in human breast cancer xenografts: mammary fat pad versus cranial tumors.

PubMed

Monsky, Wayne L; Mouta Carreira, Carla; Tsuzuki, Yoshikazu; Gohongi, Takeshi; Fukumura, Dai; Jain, Rakesh K

2002-04-01

The host microenvironment differs between primary and metastatic sites, affecting gene expression and various physiological functions. Here we show the differences in the physiological parameters between orthotopic primary and metastatic breast tumor xenografts using intravital microscopy and reveal the relationship between angiogenic gene expression and microvascular functions in vivo. ZR75-1, a human estrogen-dependent mammary carcinoma, was implanted into the mammary fat pad (primary site) of ovariectomized SCID female mice carrying estrogen pellets. The same tumor line was also grown in the cranial window (metastasis site). When tumors reached the diameter of 2.5 mm, angiogenesis, hemodynamics, and vascular permeability were measured by intravital microscopy, and expression of angiogenic growth factors was determined by quantitative reverse transcription-PCR. ZR75-1 tumors grown in the mammary fat pad had higher microvascular permeability but lower vascular density than the same tumors grown in the cranial window (2.5- and 0.7-fold, respectively). There was no significant difference in RBC velocity, vessel diameter, blood flow rate, and shear rate between two sites. The levels of vascular endothelial growth factor (VEGF), its receptors VEGFR1 and VEGFR2, and angiopoietin-1 mRNA tended to be higher in the mammary fat pad tumors than in the cranial tumors (1.5-, 1.5-, 3-, and 2-fold, respectively). The primary breast cancer exhibited higher vascular permeability, but the cranial tumor showed more angiogenesis, suggesting that the cranial environment is leakage resistant but proangiogenic. Collectively, host microenvironment is an important determinant of tumor gene expression and microvascular functions, and, thus, orthotopic breast tumor models should be useful for obtaining clinically relevant information.

Detection of altered extracellular matrix in surface layers of unstable carotid plaque: an optical spectroscopy, birefringence and microarray genetic analysis.

PubMed

Korol, Renee M; Canham, Peter B; Liu, Li; Viswanathan, Kasinath; Ferguson, Gary G; Hammond, Rob R; Finlay, Helen M; Baker, Henry V; Lopez, Cecilia; Lucas, Alexandra R

2011-01-01

Erosion and rupture of surface layers in atherosclerotic plaque can cause heart attack and stroke; however, changes in luminal surface composition are incompletely defined. Laser-induced fluorescence spectroscopy (LIFS), with limited tissue penetration, was used to investigate the surface of unstable carotid plaque and correlated with microscopy, birefringence and gene expression. Arterial matrix collagens I, III and elastin were assessed in unstable plaques (n = 25) and reference left internal mammary arteries (LIMA, n = 10). LIFS in addition to selective histological staining with picrosirius red, Movat pentachrome and immunostaining revealed decreased elastin and increased collagen I and III (P < 0.05) in carotid plaque when compared with LIMA. Within plaque, collagen I was elevated in the internal carotid region versus the common carotid region. Polarized light microscopy detected layers of aligned collagen and associated mechanical rigidity of the fibrous cap. Microarray analysis of three carotid and three LIMA specimens confirmed up-regulation of collagen I, III and IV, lysyl oxidase and MMP-12. In conclusion, LIFS analysis coupled with microscopy revealed marked regional differences in collagen I, III and elastin in surface layers of carotid plaque; indicative of plaque instability. Birefringence measurements demonstrated mechanical rigidity and weakening of the fibrous cap with complementary changes in ECM gene expression. © 2011 The Authors. Photochemistry and Photobiology © 2011 The American Society of Photobiology.

Analysis of C. elegans VIG-1 expression.

PubMed

Shin, Kyoung-Hwa; Choi, Boram; Park, Yang-Seo; Cho, Nam Jeong

2008-12-31

Double-stranded RNA (dsRNA) induces gene silencing in a sequence-specific manner by a process known as RNA interference (RNAi). The RNA-induced silencing complex (RISC) is a multi-subunit ribonucleoprotein complex that plays a key role in RNAi. VIG (Vasa intronic gene) has been identified as a component of Drosophila RISC; however, the role VIG plays in regulating RNAi is poorly understood. Here, we examined the spatial and temporal expression patterns of VIG-1, the C. elegans ortholog of Drosophila VIG, using a vig-1::gfp fusion construct. This construct contains the 908-bp region immediately upstream of vig-1 gene translation initiation site. Analysis by confocal microscopy demonstrated GFP-VIG-1 expression in a number of tissues including the pharynx, body wall muscle, hypodermis, intestine, reproductive system, and nervous system at the larval and adult stages. Furthermore, western blot analysis showed that VIG-1 is present in each developmental stage examined. To investigate regulatory sequences for vig-1 gene expression, we generated constructs containing deletions in the upstream region. It was determined that the GFP expression pattern of a deletion construct (delta-908 to -597) was generally similar to that of the non-deletion construct. In contrast, removal of a larger segment (delta-908 to -191) resulted in the loss of GFP expression in most cell types. Collectively, these results indicate that the 406-bp upstream region (-596 to -191) contains essential regulatory sequences required for VIG-1 expression.

Histidine-rich stabilized polyplexes for cMet-directed tumor-targeted gene transfer

NASA Astrophysics Data System (ADS)

Kos, Petra; Lächelt, Ulrich; Herrmann, Annika; Mickler, Frauke Martina; Döblinger, Markus; He, Dongsheng; Krhač Levačić, Ana; Morys, Stephan; Bräuchle, Christoph; Wagner, Ernst

2015-03-01

Overexpression of the hepatocyte growth factor receptor/c-Met proto oncogene on the surface of a variety of tumor cells gives an opportunity to specifically target cancerous tissues. Herein, we report the first use of c-Met as receptor for non-viral tumor-targeted gene delivery. Sequence-defined oligomers comprising the c-Met binding peptide ligand cMBP2 for targeting, a monodisperse polyethylene glycol (PEG) for polyplex surface shielding, and various cationic (oligoethanamino) amide cores containing terminal cysteines for redox-sensitive polyplex stabilization, were assembled by solid-phase supported syntheses. The resulting oligomers exhibited a greatly enhanced cellular uptake and gene transfer over non-targeted control sequences, confirming the efficacy and target-specificity of the formed polyplexes. Implementation of endosomal escape-promoting histidines in the cationic core was required for gene expression without additional endosomolytic agent. The histidine-enriched polyplexes demonstrated stability in serum as well as receptor-specific gene transfer in vivo upon intratumoral injection. The co-formulation with an analogous PEG-free cationic oligomer led to a further compaction of pDNA polyplexes with an obvious change of shape as demonstrated by transmission electron microscopy. Such compaction was critically required for efficient intravenous gene delivery which resulted in greatly enhanced, cMBP2 ligand-dependent gene expression in the distant tumor.Overexpression of the hepatocyte growth factor receptor/c-Met proto oncogene on the surface of a variety of tumor cells gives an opportunity to specifically target cancerous tissues. Herein, we report the first use of c-Met as receptor for non-viral tumor-targeted gene delivery. Sequence-defined oligomers comprising the c-Met binding peptide ligand cMBP2 for targeting, a monodisperse polyethylene glycol (PEG) for polyplex surface shielding, and various cationic (oligoethanamino) amide cores containing terminal cysteines for redox-sensitive polyplex stabilization, were assembled by solid-phase supported syntheses. The resulting oligomers exhibited a greatly enhanced cellular uptake and gene transfer over non-targeted control sequences, confirming the efficacy and target-specificity of the formed polyplexes. Implementation of endosomal escape-promoting histidines in the cationic core was required for gene expression without additional endosomolytic agent. The histidine-enriched polyplexes demonstrated stability in serum as well as receptor-specific gene transfer in vivo upon intratumoral injection. The co-formulation with an analogous PEG-free cationic oligomer led to a further compaction of pDNA polyplexes with an obvious change of shape as demonstrated by transmission electron microscopy. Such compaction was critically required for efficient intravenous gene delivery which resulted in greatly enhanced, cMBP2 ligand-dependent gene expression in the distant tumor. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr06556e

Mitochondria-targeted molecules MitoQ and SS31 reduce mutant huntingtin-induced mitochondrial toxicity and synaptic damage in Huntington's disease

PubMed Central

Yin, Xiangling; Manczak, Maria; Reddy, P. Hemachandra

2016-01-01

The objective of this study was to determine the protective effects of the mitochondria-targeted molecules MitoQ and SS31 in striatal neurons that stably express mutant huntingtin (Htt) (STHDhQ111/Q111) in Huntington's disease (HD). We studied mitochondrial and synaptic activities by measuring mRNA and the protein levels of mitochondrial and synaptic genes, mitochondrial function, and ultra-structural changes in MitoQ- and SS31-treated mutant Htt neurons relative to untreated mutant Htt neurons. We used gene expression analysis, biochemical methods, transmission electron microscopy (TEM) and confocal microscopy methods. In the MitoQ- and SS31-treated mutant Htt neurons, fission genes Drp1 and Fis1 were down-regulated, and fusion genes Mfn1, Mfn2 and Opa1 were up-regulated relative to untreated neurons, suggesting that mitochondria-targeted molecules reduce fission activity. Interestingly, the mitochondrial biogenesis genes PGC1α, PGC1β, Nrf1, Nrf2 and TFAM were up-regulated in MitoQ- and SS31-treated mutant Htt neurons. The synaptic genes synaptophysin and PSD95 were up-regulated, and mitochondrial function was normal in the MitoQ- and SS31-treated mutant Htt neurons. Immunoblotting findings of mitochondrial and synaptic proteins agreed with the mRNA findings. TEM studies revealed decreased numbers of structurally intact mitochondria in MitoQ- and SS31-treated mutant Htt neurons. These findings suggest that mitochondria-targeted molecules MitoQ and SS31 are protective against mutant Htt-induced mitochondrial and synaptic damage in HD neurons, and these mitochondria-targeted molecules are potential therapeutic molecules for the treatment of HD neurons. PMID:26908605

Mitochondria-targeted molecules MitoQ and SS31 reduce mutant huntingtin-induced mitochondrial toxicity and synaptic damage in Huntington's disease.

PubMed

Yin, Xiangling; Manczak, Maria; Reddy, P Hemachandra

2016-05-01

The objective of this study was to determine the protective effects of the mitochondria-targeted molecules MitoQ and SS31 in striatal neurons that stably express mutant huntingtin (Htt) (STHDhQ111/Q111) in Huntington's disease (HD). We studied mitochondrial and synaptic activities by measuring mRNA and the protein levels of mitochondrial and synaptic genes, mitochondrial function, and ultra-structural changes in MitoQ- and SS31-treated mutant Htt neurons relative to untreated mutant Htt neurons. We used gene expression analysis, biochemical methods, transmission electron microscopy (TEM) and confocal microscopy methods. In the MitoQ- and SS31-treated mutant Htt neurons, fission genes Drp1 and Fis1 were down-regulated, and fusion genes Mfn1, Mfn2 and Opa1 were up-regulated relative to untreated neurons, suggesting that mitochondria-targeted molecules reduce fission activity. Interestingly, the mitochondrial biogenesis genes PGC1α, PGC1β, Nrf1, Nrf2 and TFAM were up-regulated in MitoQ- and SS31-treated mutant Htt neurons. The synaptic genes synaptophysin and PSD95 were up-regulated, and mitochondrial function was normal in the MitoQ- and SS31-treated mutant Htt neurons. Immunoblotting findings of mitochondrial and synaptic proteins agreed with the mRNA findings. TEM studies revealed decreased numbers of structurally intact mitochondria in MitoQ- and SS31-treated mutant Htt neurons. These findings suggest that mitochondria-targeted molecules MitoQ and SS31 are protective against mutant Htt-induced mitochondrial and synaptic damage in HD neurons, and these mitochondria-targeted molecules are potential therapeutic molecules for the treatment of HD neurons. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Gene expression profiling of Escherichia coli in response to interactions with the lettuce rhizosphere.

PubMed

Hou, Z; Fink, R C; Black, E P; Sugawara, M; Zhang, Z; Diez-Gonzalez, F; Sadowsky, M J

2012-11-01

The objective of this study was to examine transcriptional changes in Escherichia coli when the bacterium was growing in the lettuce rhizoshpere. A combination of microarray analyses, colonization assays and confocal microscopy was used to gain a more complete understanding of bacterial genes involved in the colonization and growth of E. coli K12 in the lettuce root rhizosphere using a novel hydroponic assay system. After 3 days of interaction with lettuce roots, E. coli genes involved in protein synthesis, stress responses and attachment were up-regulated. Mutants in curli production (crl, csgA) and flagella synthesis (fliN) had a reduced capacity to attach to roots as determined by bacterial counts and by confocal laser scanning microscopy. This study indicates that E. coli K12 has the capability to colonize lettuce roots by using attachment genes and can readily adapt to the rhizosphere of lettuce plants. Results of this study show curli production and biofilm modulation genes are important for rhizosphere colonization and may provide useful targets to disrupt this process. Further studies using pathogenic strains will provide additional information about lettuce-E. coli interactions. © 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

Novel insights into the effect of nitrogen on storage protein biosynthesis and protein body development in wheat caryopsis.

PubMed

Yu, Xurun; Chen, Xinyu; Wang, Leilei; Yang, Yang; Zhu, Xiaowei; Shao, Shanshan; Cui, Wenxue; Xiong, Fei

2017-04-01

Molecular and cytological mechanisms concerning the effects of nitrogen on wheat (Triticum aestivum L.) storage protein biosynthesis and protein body development remain largely elusive. We used transcriptome sequencing, proteomics techniques, and light microscopy to investigate these issues. In total, 2585 differentially expressed genes (DEGs) and 57 differentially expressed proteins (DEPs) were found 7 days after anthesis (DAA), and 2456 DEGs and 64 DEPs were detected 18 DAA after nitrogen treatment. Gene ontology terms related to protein biosynthesis processes enriched these numbers by 678 and 582 DEGs at 7 and 18 DAA, respectively. Further, 25 Kyoto Encyclopedia of Genes and Genomes pathways were involved in protein biosynthesis at both 7 and 18 DAA. DEPs related to storage protein biosynthesis contained gliadin and glutenin subunits, most of which were up-regulated after nitrogen treatment. Quantitative real-time PCR analysis indicated that some gliadin and glutenin subunit encoding genes were differentially expressed at 18 DAA. Structural observation revealed that wheat endosperm accumulated more and larger protein bodies after nitrogen treatment. Collectively, our findings suggest that nitrogen treatment enhances storage protein content, endosperm protein body quantity, and partial processing quality by altering the expression levels of certain genes involved in protein biosynthesis pathways and storage protein expression at the proteomics level. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Association Between Germline Mutation in VSIG10L and Familial Barrett Neoplasia.

PubMed

Fecteau, Ryan E; Kong, Jianping; Kresak, Adam; Brock, Wendy; Song, Yeunjoo; Fujioka, Hisashi; Elston, Robert; Willis, Joseph E; Lynch, John P; Markowitz, Sanford D; Guda, Kishore; Chak, Amitabh

2016-10-01

Esophageal adenocarcinoma and its precursor lesion Barrett esophagus have seen a dramatic increase in incidence over the past 4 decades yet marked genetic heterogeneity of this disease has precluded advances in understanding its pathogenesis and improving treatment. To identify novel disease susceptibility variants in a familial syndrome of esophageal adenocarcinoma and Barrett esophagus, termed familial Barrett esophagus, by using high-throughput sequencing in affected individuals from a large, multigenerational family. We performed whole exome sequencing (WES) from peripheral lymphocyte DNA on 4 distant relatives from our multiplex, multigenerational familial Barrett esophagus family to identify candidate disease susceptibility variants. Gene variants were filtered, verified, and segregation analysis performed to identify a single candidate variant. Gene expression analysis was done with both quantitative real-time polymerase chain reaction and in situ RNA hybridization. A 3-dimensional organotypic cell culture model of esophageal maturation was utilized to determine the phenotypic effects of our gene variant. We used electron microscopy on esophageal mucosa from an affected family member carrying the gene variant to assess ultrastructural changes. Identification of a novel, germline disease susceptibility variant in a previously uncharacterized gene. A multiplex, multigenerational family with 14 members affected (3 members with esophageal adenocarcinoma and 11 with Barrett esophagus) was identified, and whole-exome sequencing identified a germline mutation (S631G) at a highly conserved serine residue in the uncharacterized gene VSIG10L that segregated in affected members. Transfection of S631G variant into a 3-dimensional organotypic culture model of normal esophageal squamous cells dramatically inhibited epithelial maturation compared with the wild-type. VSIG10L exhibited high expression in normal squamous esophagus with marked loss of expression in Barrett-associated lesions. Electron microscopy of squamous esophageal mucosa harboring the S631G variant revealed dilated intercellular spaces and reduced desmosomes. This study presents VSIG10L as a candidate familial Barrett esophagus susceptibility gene, with a putative role in maintaining normal esophageal homeostasis. Further research assessing VSIG10L function may reveal pathways important for esophageal maturation and the pathogenesis of Barrett esophagus and esophageal adenocarcinoma.

Association Between Germline Mutation in VSIG10L and Familial Barrett Neoplasia

PubMed Central

Fecteau, Ryan E.; Kong, Jianping; Kresak, Adam; Brock, Wendy; Song, Yeunjoo; Fujioka, Hisashi; Elston, Robert; Willis, Joseph E.; Lynch, John P.; Markowitz, Sanford D.; Guda, Kishore; Chak, Amitabh

2016-01-01

IMPORTANCE Esophageal adenocarcinoma and its precursor lesion Barrett esophagus have seen a dramatic increase in incidence over the past 4 decades yet marked genetic heterogeneity of this disease has precluded advances in understanding its pathogenesis and improving treatment. OBJECTIVE To identify novel disease susceptibility variants in a familial syndrome of esophageal adenocarcinoma and Barrett esophagus, termed familial Barrett esophagus, by using high-throughput sequencing in affected individuals from a large, multigenerational family. DESIGN, SETTING, AND PARTICIPANTS We performed whole exome sequencing (WES) from peripheral lymphocyte DNA on 4 distant relatives from our multiplex, multigenerational familial Barrett esophagus family to identify candidate disease susceptibility variants. Gene variants were filtered, verified, and segregation analysis performed to identify a single candidate variant. Gene expression analysis was done with both quantitative real-time polymerase chain reaction and in situ RNA hybridization. A 3-dimensional organotypic cell culture model of esophageal maturation was utilized to determine the phenotypic effects of our gene variant. We used electron microscopy on esophageal mucosa from an affected family member carrying the gene variant to assess ultrastructural changes. MAIN OUTCOMES AND MEASURES Identification of a novel, germline disease susceptibility variant in a previously uncharacterized gene. RESULTS A multiplex, multigenerational family with 14 members affected (3 members with esophageal adenocarcinoma and 11 with Barrett esophagus) was identified, and whole-exome sequencing identified a germline mutation (S631G) at a highly conserved serine residue in the uncharacterized gene VSIG10L that segregated in affected members. Transfection of S631G variant into a 3-dimensional organotypic culture model of normal esophageal squamous cells dramatically inhibited epithelial maturation compared with the wild-type. VSIG10L exhibited high expression in normal squamous esophagus with marked loss of expression in Barrett-associated lesions. Electron microscopy of squamous esophageal mucosa harboring the S631G variant revealed dilated intercellular spaces and reduced desmosomes. CONCLUSIONS AND RELEVANCE This study presents VSIG10L as a candidate familial Barrett esophagus susceptibility gene, with a putative role in maintaining normal esophageal homeostasis. Further research assessing VSIG10L function may reveal pathways important for esophageal maturation and the pathogenesis of Barrett esophagus and esophageal adenocarcinoma. PMID:27467440

Characterization of the Genetic Program Linked to the Development of Atrial Fibrillation in CREM-IbΔC-X Mice.

PubMed

Seidl, Matthias D; Stein, Juliane; Hamer, Sabine; Pluteanu, Florentina; Scholz, Beatrix; Wardelmann, Eva; Huge, Andreas; Witten, Anika; Stoll, Monika; Hammer, Elke; Völker, Uwe; Müller, Frank U

2017-08-01

Reduced expression of genes regulated by the transcription factors CREB/CREM (cAMP response element-binding protein/modulator) is linked to atrial fibrillation (AF) susceptibility in patients. Cardiomyocyte-directed expression of the inhibitory CREM isoform CREM-IbΔC-X in transgenic mice (TG) leads to spontaneous-onset AF preceded by atrial dilatation and conduction abnormalities. Here, we characterized the altered gene program linked to atrial remodeling and development of AF in CREM-TG mice. Atria of young (TGy, before AF onset) and old (TGo, after AF onset) TG mice were investigated by mRNA microarray profiling in comparison with age-matched wild-type controls (WTy/WTo). Proteomic alterations were profiled in young mice (8 TGy versus 8 WTy). Annotation of differentially expressed genes revealed distinct differences in biological functions and pathways before and after onset of AF. Alterations in metabolic pathways, some linked to altered peroxisome proliferator-activated receptor signaling, muscle contraction, and ion transport were already present in TGy. Electron microscopy revealed significant loss of sarcomeres and mitochondria and increased collagen and glycogen deposition in TG mice. Alterations in electrophysiological pathways became prominent in TGo, concomitant with altered gene expression of K + -channel subunits and ion channel modulators, relevant in human AF. The most prominent alterations of the gene program linked to CREM-induced atrial remodeling were identified in the expression of genes related to structure, metabolism, contractility, and electric activity regulation, suggesting that CREM transgenic mice are a valuable experimental model for human AF pathophysiology. © 2017 American Heart Association, Inc.

Hypoxia-inducible bidirectional shRNA expression vector delivery using PEI/chitosan-TBA copolymers for colorectal Cancer gene therapy.

PubMed

Javan, Bita; Atyabi, Fatemeh; Shahbazi, Majid

2018-06-01

This investigation was conducted to construct a hypoxia/colorectal dual-specific bidirectional short hairpin RNA (shRNA) expression vector and to transfect it into the colon cancer cell line HT-29 with PEI/chitosan-TBA nanoparticles for the simultaneous knock down of β-catenin and Bcl-2 under hypoxia. To construct a pRNA-bipHRE-CEA vector, the carcinoma embryonic antigen (CEA) promoter designed in two directions and the vascular endothelial growth factor (VEGF) enhancer were inserted between two promoters for hypoxic cancer specific gene expression. To confirm the therapeutic effect of the dual-specific vector, β-catenin and Bcl-2 shRNAs were inserted downstream of each promoter. The physicochemical properties, the cytotoxicity, and the transfection efficiency of these PEI/chitosan-TBA nanoparticles were investigated. In addition, the antitumor effects of the designed vector on the expression of β-catenin and Bcl-2, cell cycle distribution, and apoptosis were investigated in vitro. The silencing effect of the hypoxia-response shRNA expression vector was relatively low (18%-25%) under normoxia, whereas it was significantly increased to approximately 50%-60% in the HT-29 cell line. Moreover, the cancer cells showed significant G0/G1 arrest and increased apoptosis due to gene silencing under hypoxia. Furthermore, MTS assay, fluorescence microscopy images, and flow cytometry analyses confirmed that the PEI/chitosan-TBA blend system provided effective transfection with low cytotoxicity. This novel hypoxia-responsive shRNA expression vector may be useful for RNA interference (RNAi)-based cancer gene therapy in hypoxic colorectal tumors. Moreover, the PEI/chitosan-TBA copolymer might be a promising gene carrier for use in gene transfer in vivo. Copyright © 2018. Published by Elsevier Inc.

Rapid, high efficiency isolation of pancreatic ß-cells.

PubMed

Clardy, Susan M; Mohan, James F; Vinegoni, Claudio; Keliher, Edmund J; Iwamoto, Yoshiko; Benoist, Christophe; Mathis, Diane; Weissleder, Ralph

2015-09-02

The ability to isolate pure pancreatic ß-cells would greatly aid multiple areas of diabetes research. We developed a fluorescent exendin-4-like neopeptide conjugate for the rapid purification and isolation of functional mouse pancreatic β-cells. By targeting the glucagon-like peptide-1 receptor with the fluorescent conjugate, β-cells could be quickly isolated by flow cytometry and were >99% insulin positive. These studies were confirmed by immunostaining, microscopy and gene expression profiling on isolated cells. Gene expression profiling studies of cytofluorometrically sorted β-cells from 4 and 12 week old NOD mice provided new insights into the genetic programs at play of different stages of type-1 diabetes development. The described isolation method should have broad applicability to the β-cell field.

Nitrate-containing beetroot enhances myocyte metabolism and mitochondrial content

PubMed Central

Vaughan, Roger A.; Gannon, Nicholas P.; Carriker, Colin R.

2015-01-01

Beetroot (甜菜 tián cài) juice consumption is of current interest for improving aerobic performance by acting as a vasodilator and possibly through alterations in skeletal muscle metabolism and physiology. This work explored the effects of a commercially available beetroot supplement on metabolism, gene expression, and mitochondrial content in cultured myocytes. C2C12 myocytes were treated with various concentrations of the beetroot supplement for various durations. Glycolytic metabolism and oxidative metabolism were quantified via measurement of extracellular acidification and oxygen consumption, respectively. Metabolic gene expression was measured using quantitative reverse transcription–polymerase chain reaction, and mitochondrial content was assessed with flow cytometry and confocal microscopy. Cells treated with beetroot exhibited significantly increased oxidative metabolism, concurrently with elevated metabolic gene expression including peroxisome proliferator-activated receptor gamma coactivator-1 alpha, nuclear respiratory factor 1, mitochondrial transcription factor A, and glucose transporter 4, leading to increased mitochondrial biogenesis. Our data show that treatment with a beetroot supplement increases basal oxidative metabolism. Our observations are also among the first to demonstrate that beetroot extract is an inducer of metabolic gene expression and mitochondrial biogenesis. These observations support the need for further investigation into the therapeutic and pharmacological effects of nitrate-containing supplements for health and athletic benefits. PMID:26870674

Macromolecular Crowding Induces Spatial Correlations That Control Gene Expression Bursting Patterns

DOE PAGES

Norred, Sarah Elizabeth; Caveney, Patrick M.; Chauhan, Gaurav; ...

2018-04-24

Recent superresolution microscopy studies in E. coli demonstrate that the cytoplasm has highly variable local concentrations where macromolecular crowding plays a central role in establishing membrane-less compartmentalization. This spatial inhomogeneity significantly influences molecular transport and association processes central to gene expression. Yet, little is known about how macromolecular crowding influences gene expression bursting—the episodic process where mRNA and proteins are produced in bursts. Here, we simultaneously measured mRNA and protein reporters in cell-free systems, showing that macromolecular crowding decoupled the well-known relationship between fluctuations in the protein population (noise) and mRNA population statistics. Crowded environments led to a 10-fold increasemore » in protein noise even though there were only modest changes in the mRNA population and fluctuations. Instead, cell-like macromolecular crowding created an inhomogeneous spatial distribution of mRNA (“spatial noise”) that led to large variability in the protein production burst size. As a result, the mRNA spatial noise created large temporal fluctuations in the protein population. Furthermore, these results highlight the interplay between macromolecular crowding, spatial inhomogeneities, and the resulting dynamics of gene expression, and provide insights into using these organizational principles in both cell-based and cell-free synthetic biology.« less

Macromolecular Crowding Induces Spatial Correlations That Control Gene Expression Bursting Patterns

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

Norred, Sarah Elizabeth; Caveney, Patrick M.; Chauhan, Gaurav

Recent superresolution microscopy studies in E. coli demonstrate that the cytoplasm has highly variable local concentrations where macromolecular crowding plays a central role in establishing membrane-less compartmentalization. This spatial inhomogeneity significantly influences molecular transport and association processes central to gene expression. Yet, little is known about how macromolecular crowding influences gene expression bursting—the episodic process where mRNA and proteins are produced in bursts. Here, we simultaneously measured mRNA and protein reporters in cell-free systems, showing that macromolecular crowding decoupled the well-known relationship between fluctuations in the protein population (noise) and mRNA population statistics. Crowded environments led to a 10-fold increasemore » in protein noise even though there were only modest changes in the mRNA population and fluctuations. Instead, cell-like macromolecular crowding created an inhomogeneous spatial distribution of mRNA (“spatial noise”) that led to large variability in the protein production burst size. As a result, the mRNA spatial noise created large temporal fluctuations in the protein population. Furthermore, these results highlight the interplay between macromolecular crowding, spatial inhomogeneities, and the resulting dynamics of gene expression, and provide insights into using these organizational principles in both cell-based and cell-free synthetic biology.« less

Efficient, Glucose Responsive, and Islet-Specific Transgene Expression by a Modified Rat Insulin Promoter

PubMed Central

Chai, Renjie; Chen, Shuyuan; Ding, Jiahuan; Grayburn, Paul A

2009-01-01

This study was done to improve efficiency and islet specificity of the rat insulin promoter (RIP). Various rat insulin promoter lengths were prepared and tested in vitro to drive luciferase reporter gene expression in INS1-cells, alpha-cells, acinar cells, ductal cells, and fibroblasts. The CMV promoter was used as a positive control. In addition, the DsRed reporter gene was administered in vivo to rat pancreas by ultrasound-targeted microbubble destruction (UTMD). Confocal microscopy was used to detect the presence and distribution of DsRed within the pancreas after UTMD. A modified RIP3.1 promoter, which includes portions of the insulin gene after its transcription start site is 5-fold more active in INS-1 cells than the full length RIP promoter or the CMV promoter. RIP3.1 is regulated by glucose level and various islet transcription factors in vitro, and exhibits activity in alpha-cells, but not exocrine cells. In vivo delivery of RIP3.1-DsRed resulted in expression of DsRed protein in beta-cells, and to a lesser extent alpha cells under normal glucose conditions. No DsRed signal was present in exocrine pancreas under RIP3.1. A modified rat insulin promoter, RIP3.1, efficiently and specifically directs gene expression to endocrine pancreas. PMID:19727136

Mechanosensitive Gene Regulation by Myocardin-Related Transcription Factors is Required for Cardiomyocyte Integrity in Load-Induced Ventricular Hypertrophy.

PubMed

Trembley, Michael A; Quijada, Pearl; Agullo-Pascual, Esperanza; Tylock, Kevin M; Colpan, Mert; Dirkx, Ronald A; Myers, Jason R; Mickelsen, Deanne M; de Mesy Bentley, Karen; Rothenberg, Eli; Moravec, Christine S; Alexis, Jeffrey D; Gregorio, Carol C; Dirksen, Robert T; Delmar, Mario; Small, Eric M

2018-05-01

Background -Hypertrophic cardiomyocyte (CM) growth and dysfunction accompanies various forms of heart disease. The mechanisms responsible for transcriptional changes that impact cardiac physiology and the transition to heart failure (HF) are not well understood. The intercalated disc (ID) is a specialized intercellular junction coupling CM electrical activity and force transmission, and is gaining attention as a mechanosensitive signaling hub and hotspot for causative mutations in cardiomyopathy. Methods -Transmission electron microscopy, confocal microscopy, and single-molecule localization microscopy (SMLM) were used to examine changes in ID structure and protein localization in the murine and human heart. We conducted detailed cardiac functional assessment and transcriptional profiling of mice lacking myocardin-related transcription factor-A (MRTF-A) and -B specifically in adult CMs to evaluate the role of mechanosensitive regulation of gene expression in load-induced ventricular remodeling. Results -We found that MRTFs localize to IDs in the healthy human heart and accumulate in the nucleus in heart failure (HF). Although mice lacking MRTFs in adult CMs display normal cardiac physiology at baseline, pressure overload leads to rapid HF characterized by sarcomere disarray, ID disintegration, chamber dilation and wall thinning, cardiac functional decline, and partially penetrant acute lethality. Transcriptional profiling reveals a program of actin cytoskeleton and CM adhesion genes driven by MRTFs during pressure overload. Indeed, conspicuous remodeling of gap junctions at IDs identified by SMLM may partially stem from a reduction in Mapre1 expression, which we show is a direct mechanosensitive MRTF target. Conclusions -Taken together, our study describes a novel paradigm in which MRTFs control an acute mechanosensitive signaling circuit that coordinates crosstalk between the actin and microtubule cytoskeleton and maintains ID integrity and CM homeostasis in heart disease.

[Zinc-dependent metalloprotease 1 promotes apoptosis of RAW264.7 macrophages].

PubMed

Li, Peng; He, Yonglin; Zhang, Jiming; Fang, Chencheng

2015-12-01

To construct the eukaryotic expression vector of zinc-dependent metalloprotease 1 (zmp1) gene from Bacillus Calmette-Guerin (BCG) and investigate its impact on the apoptosis of RAW264.7 macrophages. Zmp1 gene was amplified from the genome of BCG by PCR. The zmp1 gene fragment was inserted into multiple cloning sites of pEGFP-N1 to construct the eukaryotic expression vector pEGFP-N1-zmp1. The constructed pEGFP-N1-zmp1 was transfected into RAW264.7 cells by Lipofectamine(TM) 2000. The expression of green fluorescent protein (GFP) was observed by fluorescence microscopy. The zmp1 mRNA was detected by quantitative real-time PCR (qR-PCR). The effect of Zmp1 protein on the apoptosis of RAW264.7 macrophages was detected by flow cytometry (FCM). With zmp1 gene amplified by PCR, we successfully constructed the recombinant vector pEGFP-N1-zmp1 as demonstrated by restriction enzyme analysis and sequencing. GFP was seen in RAW264.7 cells 24 hours after transfected with the recombinant plasmid. As qRT-PCR showed, the expression level of zmp1 mRNA was up-regulated. The early apoptotic rate increased 48 hours after transfection. The increased expression of Zmp1 in RAW264.7 cells promotes the apoptosis of RAW264.7 cells.

Cytotoxicity and expression of genes involved in the cellular stress response and apoptosis in mammalian fibroblast exposed to cotton cellulose nanofibers

NASA Astrophysics Data System (ADS)

Pereira, M. M.; Raposo, N. R. B.; Brayner, R.; Teixeira, E. M.; Oliveira, V.; Quintão, C. C. R.; Camargo, L. S. A.; Mattoso, L. H. C.; Brandão, H. M.

2013-02-01

Cellulose nanofibers (CNF) have mechanical properties that make them very attractive for applications in the construction of polymeric matrices, drug delivery and tissue engineering. However, little is known about their impact on mammalian cells. The objective of this study was to evaluate the cytotoxicity of CNF and their effect on gene expression of fibroblasts cultured in vitro. The morphology of CNF was analyzed by transmission electron microscopy and the surface charge by Zeta potential. Cell viability was analyzed by flow cytometry assay and gene expression of biomarkers focused on cell stress response such as Heat shock protein 70.1 (HSP70.1) and Peroxiredoxin 1 (PRDX1) and apoptosis as B-cell leukemia (BCL-2) and BCL-2 associated X protein (BAX) by RT-PCR assay. Low concentrations of CNF (0.02-100 μg ml-1) did not cause cell death; however, at concentrations above 200 μg ml-1, the nanofibers significantly decreased cell viability (86.41 ± 5.37%). The exposure to high concentrations of CNF (2000 and 5000 μg ml-1) resulted in increased HSP70.1, PRDX1 and BAX gene expression. The current study concludes that, under the conditions tested, high concentrations (2000 and 5000 μg ml-1) of CNF cause decreased cell viability and affect the expression of stress- and apoptosis-associated molecular markers.

Pinoresinol reductase 1 impacts lignin distribution during secondary cell wall biosynthesis in Arabidopsis

DOE PAGES

Zhao, Qiao; Zeng, Yining; Yin, Yanbin; ...

2014-08-05

In this paper, pinoresinol reductase (PrR) catalyzes the conversion of the lignan (-)-pinoresinol to (-)-lariciresinol in Arabidopsis thaliana, where it is encoded by two genes, PrR1 and PrR2, that appear to act redundantly. PrR1 is highly expressed in lignified inflorescence stem tissue, whereas PrR2 expression is barely detectable in stems. Co-expression analysis has indicated that PrR1 is co-expressed with many characterized genes involved in secondary cell wall biosynthesis, whereas PrR2 expression clusters with a different set of genes. The promoter of the PrR1 gene is regulated by the secondary cell wall related transcription factors SND1 and MYB46. The loss-of-function mutantmore » of PrR1 shows, in addition to elevated levels of pinoresinol, significantly decreased lignin content and a slightly altered lignin structure with lower abundance of cinnamyl alcohol end groups. Stimulated Raman scattering (SRS) microscopy analysis indicated that the lignin content of the prr1-1 loss-of-function mutant is similar to that of wild-type plants in xylem cells, which exhibit a normal phenotype, but is reduced in the fiber cells. Finally, together, these data suggest an association of the lignan biosynthetic enzyme encoded by PrR1 with secondary cell wall biosynthesis in fiber cells.« less

Automated solid-phase subcloning based on beads brought into proximity by magnetic force.

PubMed

Hudson, Elton P; Nikoshkov, Andrej; Uhlen, Mathias; Rockberg, Johan

2012-01-01

In the fields of proteomics, metabolic engineering and synthetic biology there is a need for high-throughput and reliable cloning methods to facilitate construction of expression vectors and genetic pathways. Here, we describe a new approach for solid-phase cloning in which both the vector and the gene are immobilized to separate paramagnetic beads and brought into proximity by magnetic force. Ligation events were directly evaluated using fluorescent-based microscopy and flow cytometry. The highest ligation efficiencies were obtained when gene- and vector-coated beads were brought into close contact by application of a magnet during the ligation step. An automated procedure was developed using a laboratory workstation to transfer genes into various expression vectors and more than 95% correct clones were obtained in a number of various applications. The method presented here is suitable for efficient subcloning in an automated manner to rapidly generate a large number of gene constructs in various vectors intended for high throughput applications.

Automated Solid-Phase Subcloning Based on Beads Brought into Proximity by Magnetic Force

PubMed Central

Hudson, Elton P.; Nikoshkov, Andrej; Uhlen, Mathias; Rockberg, Johan

2012-01-01

In the fields of proteomics, metabolic engineering and synthetic biology there is a need for high-throughput and reliable cloning methods to facilitate construction of expression vectors and genetic pathways. Here, we describe a new approach for solid-phase cloning in which both the vector and the gene are immobilized to separate paramagnetic beads and brought into proximity by magnetic force. Ligation events were directly evaluated using fluorescent-based microscopy and flow cytometry. The highest ligation efficiencies were obtained when gene- and vector-coated beads were brought into close contact by application of a magnet during the ligation step. An automated procedure was developed using a laboratory workstation to transfer genes into various expression vectors and more than 95% correct clones were obtained in a number of various applications. The method presented here is suitable for efficient subcloning in an automated manner to rapidly generate a large number of gene constructs in various vectors intended for high throughput applications. PMID:22624028

Metabolic pathway engineering in cotton: Biosynthesis of polyhydroxybutyrate in fiber cells

PubMed Central

John, Maliyakal E.; Keller, Greg

1996-01-01

Alcaligenes eutrophus genes encoding the enzymes, β-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB), and polyhydroxyalkanoate synthase (phaC) catalyze the production of aliphatic polyester poly-d-(−)-3-hydroxybutyrate (PHB) from acetyl-CoA. PHB is a thermoplastic polymer that may modify fiber properties when synthesized in cotton. Endogenous β-ketothiolase activity is present in cotton fibers. Hence cotton was transformed with engineered phaB and phaC genes by particle bombardment, and transgenic plants were selected based on marker gene, β-glucuronidase (GUS), expression. Fibers of 10 transgenic plants expressed phaB gene, while eight plants expressed both phaB and phaC genes. Electron microscopy examination of fibers expressing both genes indicated the presence of electron-lucent granules in the cytoplasm. High pressure liquid chromatography, gas chromatography, and mass spectrometry evidence suggested that the new polymer produced in transgenic fibers is PHB. Sixty-six percent of the PHB in fibers is in the molecular mass range of 0.6 × 106 to 1.8 × 106 Da. The presence of PHB granules in transgenic fibers resulted in measurable changes of thermal properties. The fibers exhibited better insulating characteristics. The rate of heat uptake and cooling was slower in transgenic fibers, resulting in higher heat capacity. These data show that metabolic pathway engineering in cotton may enhance fiber properties by incorporating new traits from other genetic sources. This is an important step toward producing new generation fibers for the textile industry. PMID:11038522

Cross-talk among HMGA1 and FoxO1 in control of nuclear insulin signaling.

PubMed

Chiefari, Eusebio; Arcidiacono, Biagio; Palmieri, Camillo; Corigliano, Domenica Maria; Morittu, Valeria Maria; Britti, Domenico; Armoni, Michal; Foti, Daniela Patrizia; Brunetti, Antonio

2018-06-04

As a mediator of insulin-regulated gene expression, the FoxO1 transcription factor represents a master regulator of liver glucose metabolism. We previously reported that the high-mobility group AT-hook 1 (HMGA1) protein, a molecular switch for the insulin receptor gene, functions also as a downstream target of the insulin receptor signaling pathway, representing a critical nuclear mediator of insulin function. Here, we investigated whether a functional relationship existed between FoxO1 and HMGA1, which might help explain insulin-mediated gene transcription in the liver. To this end, as a model study, we investigated the canonical FoxO1-HMGA1-responsive IGFBP1 gene, whose hepatic expression is regulated by insulin. By using a conventional GST-pull down assay combined with co-immunoprecipitation and Fluorescence Resonance Energy Transfer (FRET) analyses, we provide evidence of a physical interaction between FoxO1 and HMGA1. Further investigation with chromatin immunoprecipitation, confocal microscopy, and Fluorescence Recovery After Photobleaching (FRAP) technology indicated a functional significance of this interaction, in both basal and insulin-stimulated states, providing evidence that, by modulating FoxO1 transactivation, HMGA1 is essential for FoxO1-induced IGFBP1 gene expression, and thereby a critical modulator of insulin-mediated FoxO1 regulation in the liver. Collectively, our findings highlight a novel FoxO1/HMGA1-mediated mechanism by which insulin may regulate gene expression and metabolism.

Visualization of glucagon secretion from pancreatic α cells by bioluminescence video microscopy: Identification of secretion sites in the intercellular contact regions

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

Yokawa, Satoru; School of Pharmacy, Aichi Gakuin University, Nagoya 464-8650; Suzuki, Takahiro

We have firstly visualized glucagon secretion using a method of video-rate bioluminescence imaging. The fusion protein of proglucagon and Gaussia luciferase (PGCG-GLase) was used as a reporter to detect glucagon secretion and was efficiently expressed in mouse pancreatic α cells (αTC1.6) using a preferred human codon-optimized gene. In the culture medium of the cells expressing PGCG-GLase, luminescence activity determined with a luminometer was increased with low glucose stimulation and KCl-induced depolarization, as observed for glucagon secretion. From immunochemical analyses, PGCG-GLase stably expressed in clonal αTC1.6 cells was correctly processed and released by secretory granules. Luminescence signals of the secreted PGCG-GLase frommore » the stable cells were visualized by video-rate bioluminescence microscopy. The video images showed an increase in glucagon secretion from clustered cells in response to stimulation by KCl. The secretory events were observed frequently at the intercellular contact regions. Thus, the localization and frequency of glucagon secretion might be regulated by cell-cell adhesion. - Highlights: • The fused protein of proglucagon to Gaussia luciferase was used as a reporter. • The fusion protein was highly expressed using a preferred human-codon optimized gene. • Glucagon secretion stimulated by depolarization was determined by luminescence. • Glucagon secretion in α cells was visualized by bioluminescence imaging. • Glucagon secretion sites were localized in the intercellular contact regions.« less

Imbalance of caveolin-1 and eNOS expression in the pulmonary vasculature of experimental diaphragmatic hernia.

PubMed

Hofmann, Alejandro; Gosemann, Jan-Hendrik; Takahashi, Toshiaki; Friedmacher, Florian; Duess, Johannes W; Puri, Prem

2014-08-01

Caveolin-1 (Cav-1) exerts major regulatory functions on intracellular signaling pathways originating at the plasma membrane. Cav-1 is a key regulator in adverse lung remodeling and the development of pulmonary hypertension (PH) regulating vasomotor tone through its ability to reduce nitric oxide (NO) production. This low-output endothelial NO synthase (eNOS) derived NO maintains normal pulmonary vascular homeostasis. Cav-1 deficiency leads to increased bioavailability of NO, which has been linked to increased nitrosative stress. Inhibition of eNOS reduced oxidant production and reversed PH, supporting the concept that Cav-1 regulation of eNOS activity is crucial to endothelial homeostasis in lungs. We designed this study to investigate the hypothesis that expression of Cav-1 is downregulated while eNOS expression is upregulated by the pulmonary endothelium in the nitrofen-induced congenital diaphragmatic hernia (CDH). Pregnant rats were exposed to nitrofen or vehicle on day 9.5 (D9.5). Fetuses were sacrificed on D21 and divided into nitrofen and control groups. Quantitative real-time polymerase chain reaction, Western blotting, and confocal immunofluorescence were performed to determine pulmonary gene expression levels and protein expression of Cav-1 and eNOS. Pulmonary Cav-1 gene expression levels were significantly decreased, while eNOS gene expression was significantly increased in nitrofen-induced CDH(+). Western blotting and confocal microscopy revealed decreased pulmonary Cav-1 protein expression, while eNOS protein expression was increased in CDH(+) compared to controls. The striking evidence of markedly decreased gene and protein expression of Cav-1 with concurrently increased eNOS gene and protein expression in the pulmonary vasculature suggests that activation of eNOS secondary to Cav-1 deficiency may play an important role in the pathogenesis of PH in the nitrofen-induced CDH. © 2014 Wiley Periodicals, Inc.

Construction of a laccase chimerical gene: recombinant protein characterization and gene expression via yeast surface display.

PubMed

Bleve, G; Lezzi, C; Spagnolo, S; Rampino, P; Perrotta, C; Mita, G; Grieco, Francesco

2014-03-01

The ERY4 laccase gene from Pleurotus eryngii was expressed in Saccharomyces cerevisiae and the recombinant laccase resulted to be not biologically active. This gene was thus modified to obtain chimerical enzymes derived from the substitution of N-, C- and both N- and C-terminal regions with the corresponding regions of Ery3 laccase, another laccase isoform of P. eryngii. The chimerical isoform named 4NC3, derived from the substitution of both N- and C-terminal regions, showed the best performances in terms of enzymatic activities, affinities for different substrates and stability at a broad range of temperatures and pHs. The chimerical 4NC3 laccase isoform was displayed on the cell surface of S. cerevisiae using the N-terminal fusion with either the Pir2 or the Flo1 S. cerevisiae proteins as anchor attachment sequence. Immunofluorescence microscopy and Western blot analyses confirmed the localization of 4NC3 on the yeast cell surface. The enzyme activity on specific laccase substrates revealed that 4NC3 laccase was immobilized in active form on the cell surface. To our knowledge, this is the first example of expression of a chimerical fungal laccase by yeast cell display.

Hairy Root Transformation Using Agrobacterium rhizogenes as a Tool for Exploring Cell Type-Specific Gene Expression and Function Using Tomato as a Model1[W][OPEN

PubMed Central

Ron, Mily; Kajala, Kaisa; Pauluzzi, Germain; Wang, Dongxue; Reynoso, Mauricio A.; Zumstein, Kristina; Garcha, Jasmine; Winte, Sonja; Masson, Helen; Inagaki, Soichi; Federici, Fernán; Sinha, Neelima; Deal, Roger B.; Bailey-Serres, Julia; Brady, Siobhan M.

2014-01-01

Agrobacterium rhizogenes (or Rhizobium rhizogenes) is able to transform plant genomes and induce the production of hairy roots. We describe the use of A. rhizogenes in tomato (Solanum spp.) to rapidly assess gene expression and function. Gene expression of reporters is indistinguishable in plants transformed by Agrobacterium tumefaciens as compared with A. rhizogenes. A root cell type- and tissue-specific promoter resource has been generated for domesticated and wild tomato (Solanum lycopersicum and Solanum pennellii, respectively) using these approaches. Imaging of tomato roots using A. rhizogenes coupled with laser scanning confocal microscopy is facilitated by the use of a membrane-tagged protein fused to a red fluorescent protein marker present in binary vectors. Tomato-optimized isolation of nuclei tagged in specific cell types and translating ribosome affinity purification binary vectors were generated and used to monitor associated messenger RNA abundance or chromatin modification. Finally, transcriptional reporters, translational reporters, and clustered regularly interspaced short palindromic repeats-associated nuclease9 genome editing demonstrate that SHORT-ROOT and SCARECROW gene function is conserved between Arabidopsis (Arabidopsis thaliana) and tomato. PMID:24868032

Improving genetic immobilization of a cellulase on yeast cell surface for bioethanol production using cellulose.

PubMed

Yang, Jinying; Dang, Hongyue; Lu, Jian Ren

2013-04-01

In this study, Saccharomyces cerevisiae was genetically engineered to harbor the capability of utilizing celluloses for bioethanol production by displaying active cellulolytic enzymes on the cell surface. An endo-1,4-β-glucanase gene egX was cloned from Bacillus pumilus C-9 and its expression products, the EGX cellulases, were displayed on the cell surface of S. cerevisiae by fusing egX with aga2 that encodes the binding subunit of the S. cerevisiae cell wall protein α-agglutinin. To achieve high gene copies and stability, multicopy integration was obtained by integrating the fusion aga2-egX gene into the rDNA region of the S. cerevisiae chromosome. To achieve high expression and surface display efficiency, the aga2-egX gene was expressed under the control of a strong promoter. The presence of the enzymatically active cellulase fusion proteins on the S. cerevisiae cell surface was verified by carboxymethyl cellulase activity assay and immunofluorescence microscopy. This work presented a promising strategy to genetically engineer yeasts to perform efficient fermentation of cellulosic materials for bioethanol production. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Expression of tomato prosystemin gene in Arabidopsis reveals systemic translocation of its mRNA and confers necrotrophic fungal resistance.

PubMed

Zhang, Haiyan; Yu, Pengli; Zhao, Jiuhai; Jiang, Hongling; Wang, Haiyang; Zhu, Yingfang; Botella, Miguel A; Šamaj, Jozef; Li, Chuanyou; Lin, Jinxing

2018-01-01

Systemin (SYS), an octadecapeptide hormone processed from a 200-amino-acid precursor (prosystemin, PS), plays a central role in the systemic activation of defense genes in tomato in response to herbivore and pathogen attacks. However, whether PS mRNA is transferable and its role in systemic defense responses remain unknown. We created the transgenic tomato PS gene tagged with the green fluorescent protein (PS-GFP) using a shoot- or root-specific promoter, and the constitutive 35S promoter in Arabidopsis. Subcellular localization of PS-/SYS-GFP was observed using confocal laser scanning microscopy and gene transcripts were determined using quantitative real-time PCR. In Arabidopsis, PS protein can be processed and SYS is secreted. Shoot-/root-specific expression of PS-GFP in Arabidopsis, and grafting experiments, revealed that the PS mRNA moves in a bi-directional manner. We also found that ectopic expression of PS improves Arabidopsis resistance to the necrotrophic fungus Botrytis cinerea, consistent with substantial upregulation of the transcript levels of specific pathogen-responsive genes. Our results provide novel insights into the multifaceted mechanism of SYS signaling transport and its potential application in genetic engineering for increasing pathogen resistance across diverse plant families. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Visual detection of multidrug resistance gene in living cell using the molecular beacon imaging

NASA Astrophysics Data System (ADS)

Zhou, Qiumei; Ma, Yi; Gu, Yueqing

2014-09-01

A major problem in cancer treatment is the development of resistance to chemotherapeutic agents in tumor cells. Detection of effective prognostic biomarkers and targets are of crucial importance to the management of individualized therapies. However, quantitative analysis of the drug resistance gene had been difficult because of technical limitations. In this study, we designed and used a special hairpin deoxyribonucleic acid (DNA), which served as a beacon for detecting human drug resistance indicater. Upon hybridizing with the target mRNA, the hairpin DNA modified gold nanoparticle beacons (hDAuNP beacons) release the fluorophores attached at 5'end of the oligonucleotide sequence. The fluorescence properties of the beacon before and after the hybridization with the complementary DNA were confirmed in vitro. The hDAuNP beacons could be taken up by living cells with low inherent cytotoxicity and higher stability. hDAuNP beacon imaged by confocal laser scanning microscopy to detect the resistance gene expression. The detected fluorescence in MCF7and MCF7/ADR cells correlates with the specific drug resistance gene expression, which is consistent with the result from Q-PCR. Thus, this approach overcame many of the challenges of previous techniques by creating highly sensitive and effective intracellular probes for monitoring gene expression.

Imbalance of NFATc2 and KV1.5 Expression in Rat Pulmonary Vasculature of Nitrofen-Induced Congenital Diaphragmatic Hernia.

PubMed

Zimmer, Julia; Takahashi, Toshiaki; Hofmann, Alejandro Daniel; Puri, Prem

2017-02-01

Aim of the Study  Nuclear factor of activated T-cell (NFATc2), a Ca 2+ /calcineurin-dependent transcription factor, is reported to be activated in human and animal pulmonary hypertension (PH). KV1.5, a voltage-gated K + (KV) channel, is expressed in pulmonary artery smooth muscle cells (PASMC) and downregulated in PASMC in patients and animals with PH. Furthermore, activation of NFATc2 downregulates expression of KV1.5 channels, leading to excessive PASMC proliferation. The aim of this study was to investigate the pulmonary vascular expression of NFATc2 and KV1.5 in rats with nitrofen-induced congenital diaphragmatic hernia (CDH). Materials and Methods  After ethical approval, time-pregnant Sprague-Dawley rats received nitrofen or vehicle on gestational day 9 (D9). When sacrificed on D21, the fetuses ( n  = 22) were divided into CDH and control groups. Using quantitative real-time polymerase chain reaction and western blotting, we determined the gene and protein expression of NFATc2 and KV1.5. Confocal microscopy was used to detect both proteins in the pulmonary vasculature. Results  Relative mRNA levels of NFATc2 were significantly upregulated and KV1.5 levels were significantly downregulated in CDH lungs compared with controls ( p  < 0.05). Western blotting confirmed the imbalanced pulmonary protein expression of both proteins. An increased pulmonary vascular expression of NFATc2 and a diminished expression of KV1.5 in CDH lungs compared with controls were seen in confocal microscopy. Conclusions  This study demonstrates for the first time an altered gene and protein expression of NFATc2 and KV1.5 in the pulmonary vasculature of nitrofen-induced CDH. Upregulation of NFATc2 with concomitant downregulation of KV1.5 channels may contribute to abnormal vascular remodeling resulting in PH in this model. Georg Thieme Verlag KG Stuttgart · New York.

SPONTANEOUS AND MNNG-INDUCED REVERSION OF AN EGFP CONSTRUCT IN HELA CELLS: AN ASSAY FOR OBSERVING MUTATIONS IN LIVING CELLS BY FLUORESCENT MICROSCOPY

EPA Science Inventory

A HeLa cell line stably expressing the Enhanced Green Fluorescence Protein (EGFP) gene, interrupted by the IVS2-654 intron, was studied without treatment and after treatment with a single standard dose of 15 ?M of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). This assay was done ...

The genome-wide expression profile of Curcuma longa-treated cisplatin-stimulated HEK293 cells

PubMed Central

Sohn, Sung-Hwa; Ko, Eunjung; Chung, Hwan-Suck; Lee, Eun-Young; Kim, Sung-Hoon; Shin, Minkyu; Hong, Moochang; Bae, Hyunsu

2010-01-01

AIM The rhizome of turmeric, Curcuma longa (CL), is a herbal medicine used in many traditional prescriptions. It has previously been shown that CL treatment showed greater than 47% recovery from cisplatin-induced cell damage in human kidney HEK 293 cells. This study was conducted to evaluate the recovery mechanisms of CL that occur during cisplatin induced nephrotoxicity by examining the genome wide mRNA expression profiles of HEK 293 -cells. METHOD Recovery mechanisms of CL that occur during cisplatin-induced nephrotoxicity were determined by microarray, real-time PCR, immunofluorescent confocal microscopy and Western blot analysis. RESULTS The results of microarray analysis and real-time PCR revealed that NFκB pathway-related genes and apoptosis-related genes were down-regulated in CL-treated HEK 293 cells. In addition, immunofluorescent confocal microscopy and Western blot analysis revealed that NFκB p65 nuclear translocation was inhibited in CL-treated HEK 293 cells. Therefore, the mechanism responsible for the effects of CL on HEK 293 cells is closely associated with regulation of the NFκB pathway. CONCLUSION CL possesses novel therapeutic agents that can be used for the prevention or treatment of cisplatin-induced renal disorders. PMID:20840446

The dam replacing gene product enhances Neisseria gonorrhoeae FA1090 viability and biofilm formation

PubMed Central

Kwiatek, Agnieszka; Bacal, Pawel; Wasiluk, Adrian; Trybunko, Anastasiya; Adamczyk-Poplawska, Monika

2014-01-01

Many Neisseriaceae do not exhibit Dam methyltransferase activity and, instead of the dam gene, possess drg (dam replacing gene) inserted in the leuS/dam locus. The drg locus in Neisseria gonorrhoeae FA1090 has a lower GC-pairs content (40.5%) compared to the whole genome of N. gonorrhoeae FA1090 (52%). The gonococcal drg gene encodes a DNA endonuclease Drg, with GmeATC specificity. Disruption of drg or insertion of the dam gene in gonococcal genome changes the level of expression of genes as shown by transcriptome analysis. For the drg-deficient N. gonorrhoeae mutant, a total of 195 (8.94% of the total gene pool) genes exhibited an altered expression compared to the wt strain by at least 1.5 fold. In dam-expressing N. gonorrhoeae mutant, the expression of 240 genes (11% of total genes) was deregulated. Most of these deregulated genes were involved in translation, DNA repair, membrane biogenesis and energy production as shown by cluster of orthologous group analysis. In vivo, the inactivation of drg gene causes the decrease of the number of live neisserial cells and long lag phase of growth. The insertion of dam gene instead of drg locus restores cell viability. We have also shown that presence of the drg gene product is important for N. gonorrhoeae FA1090 in adhesion, including human epithelial cells, and biofilm formation. Biofilm produced by drg-deficient strain is formed by more dispersed cells, compared to this one formed by parental strain as shown by scanning electron and confocal microscopy. Also adherence assays show a significantly smaller biomass of formed biofilm (OD570 = 0.242 ± 0.038) for drg-deficient strain, compared to wild-type strain (OD570 = 0.378 ± 0.057). Dam-expressing gonococcal cells produce slightly weaker biofilm with cells embedded in an extracellular matrix. This strain has also a five times reduced ability for adhesion to human epithelial cells. In this context, the presence of Drg is more advantageous for N. gonorrhoeae biology than Dam presence. PMID:25566225

Mucosal Transcriptomics Implicates Under Expression of BRINP3 in the Pathogenesis of Ulcerative Colitis

PubMed Central

Smith, Philip J.; Levine, Adam P.; Dunne, Jenny; Guilhamon, Paul; Turmaine, Mark; Sewell, Gavin W.; O'Shea, Nuala R.; Vega, Roser; Paterson, Jennifer C.; Oukrif, Dahmane; Beck, Stephan; Bloom, Stuart L.; Novelli, Marco; Rodriguez-Justo, Manuel; Smith, Andrew M.

2014-01-01

Background: Mucosal abnormalities are potentially important in the primary pathogenesis of ulcerative colitis (UC). We investigated the mucosal transcriptomic expression profiles of biopsies from patients with UC and healthy controls, taken from macroscopically noninflamed tissue from the terminal ileum and 3 colonic locations with the objective of identifying abnormal molecules that might be involved in disease development. Methods: Whole-genome transcriptional analysis was performed on intestinal biopsies taken from 24 patients with UC, 26 healthy controls, and 14 patients with Crohn's disease. Differential gene expression analysis was performed at each tissue location separately, and results were then meta-analyzed. Significantly, differentially expressed genes were validated using quantitative polymerase chain reaction. The location of gene expression within the colon was determined using immunohistochemistry, subcellular fractionation, electron and confocal microscopy. DNA methylation was quantified by pyrosequencing. Results: Only 4 probes were abnormally expressed throughout the colon in patients with UC with Bone morphogenetic protein/Retinoic acid Inducible Neural-specific 3 (BRINP3) being the most significantly underexpressed. Attenuated expression of BRINP3 in UC was independent of current inflammation, unrelated to phenotype or treatment, and remained low at rebiopsy an average of 22 months later. BRINP3 is localized to the brush border of the colonic epithelium and expression is influenced by DNA methylation within its promoter. Conclusions: Genome-wide expression analysis of noninflamed mucosal biopsies from patients with UC identified BRINP3 as significantly underexpressed throughout the colon in a large subset of patients with UC. Low levels of this gene could predispose or contribute to the maintenance of the characteristic mucosal inflammation seen in this condition. PMID:25171508

The effect of collagen coating on titanium with nanotopography on in vitro osteogenesis.

PubMed

Costa, Daniel G; Ferraz, Emanuela P; Abuna, Rodrigo P F; de Oliveira, Paulo T; Morra, Marco; Beloti, Marcio M; Rosa, Adalberto L

2017-10-01

Several studies have shown the positive effects of Ti either with nanotopography or coated with collagen on osteoblast differentiation. Thus, we hypothesized that the association of nanotopography with collagen may increase the in vitro osteogenesis on Ti surface. Ti discs with nanotopography with or without collagen coating were characterized by scanning electron microscopy and atomic force microscopy. Rat calvaria-derived osteoblastic cells were cultured on both Ti surfaces for up to 14 days and the following parameters were evaluated: cell proliferation, alkaline phosphatase (ALP) activity, extracellular matrix mineralization, protein expression of bone sialoprotein (BSP) and osteopontin (OPN), and gene expression of collagen type 1a (Coll1a), runt-related transcription factor 2 (Runx2), osterix (OSX), osteocalcin (OC), Ki67, Survivin, and Bcl2-associated X protein (BAX). Surface characterization evidenced that collagen coating did not alter the nanotopography. Collagen coating increased cell proliferation, ALP activity, extracellular matrix mineralization, and Coll1a, OSX, OC, and BAX gene expression. Also, OPN and BSP proteins were strongly detected in cultures grown on both Ti surfaces. In conclusion, our results showed that the combination of nanotopography with collagen coating stimulates the early, intermediate, and final events of the in vitro osteogenesis and may be considered a potential approach to promote osseointegration of Ti implants. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2783-2788, 2017. © 2017 Wiley Periodicals, Inc.

Disease-Causing Mutations in BEST1 Gene Are Associated with Altered Sorting of Bestrophin-1 Protein

PubMed Central

Doumanov, Jordan A.; Zeitz, Christina; Gimenez, Paloma Dominguez; Audo, Isabelle; Krishna, Abhay; Alfano, Giovanna; Diaz, Maria Luz Bellido; Moskova-Doumanova, Veselina; Lancelot, Marie-Elise; Sahel, José-Alain; Nandrot, Emeline F.; Bhattacharya, Shomi S.

2013-01-01

Mutations in BEST1 gene, encoding the bestrophin-1 (Best1) protein are associated with macular dystrophies. Best1 is predominantly expressed in the retinal pigment epithelium (RPE), and is inserted in its basolateral membrane. We investigated the cellular localization in polarized MDCKII cells of disease-associated Best1 mutant proteins to study specific sorting motifs of Best1. Real-time PCR and western blots for endogenous expression of BEST1 in MDCK cells were performed. Best1 mutant constructs were generated using site-directed mutagenesis and transfected in MDCK cells. For protein sorting, confocal microscopy studies, biotinylation assays and statistical methods for quantification of mislocalization were used. Analysis of endogenous expression of BEST1 in MDCK cells revealed the presence of BEST1 transcript but no protein. Confocal microscopy and quantitative analyses indicate that transfected normal human Best1 displays a basolateral localization in MDCK cells, while cell sorting of several Best1 mutants (Y85H, Q96R, L100R, Y227N, Y227E) was altered. In contrast to constitutively active Y227E, constitutively inactive Y227F Best1 mutant localized basolaterally similar to the normal Best1 protein. Our data suggest that at least three basolateral sorting motifs might be implicated in proper Best1 basolateral localization. In addition, non-phosphorylated tyrosine 227 could play a role for basolateral delivery. PMID:23880862

Expression Profiles of TGF-β and TLR Pathways in Porphyromonas gingivalis and Prevotella intermedia Challenged Osteoblasts.

PubMed

Aydin, Kubra; Ekinci, Fatma Yesim; Korachi, May

2015-04-01

The presence of certain oral pathogens at implant sites can hinder the osseointegration process. However, it is unclear how and by what microorganisms it happens. This study investigated whether the presence of oral pathogens of Porphyromonas gingivalis and Prevotella intermedia individually, play a role in the failure of bone formation by determining the expression profiles of Transforming Growth Factor Beta (TGF-β/Bone Morphogenic Protein (BMP) and Toll-Like Receptor (TLR) pathways in challenged osteoblasts. Cell viability of P. gingivalis and P. intermedia challenged osteoblasts were determined by WST assay. Changes in osteoblast morphology and inhibition of mineralization were observed by Scanning Electron Microscopy (SEM) and Von Kossa staining, respectively. Expression of TGF-β and TLR pathway genes on challenged cells were identified by RT profiler array. Both P. gingivalis and P. intermedia challenges resulted in reduced viability and mineralization of osteoblasts. Viability was reduced to 56.8% (P. gingivalis) and 52.75% (P. intermedia) at 1000 multiplicity. Amongst 48 genes examined, expressions of BMPER, SMAD1, IL8 and NFRKB were found to be highly upregulated by both bacterial challenges (Fold Change > 4). P. gingivalis and P. intermedia could play a role in implant failure by changing the expression profiles of genes related to bone formation and resorption.

Identification and expression analysis of ERF transcription factor genes in petunia during flower senescence and in response to hormone treatments.

PubMed

Liu, Juanxu; Li, Jingyu; Wang, Huinan; Fu, Zhaodi; Liu, Juan; Yu, Yixun

2011-01-01

Ethylene-responsive element-binding factor (ERF) genes constitute one of the largest transcription factor gene families in plants. In Arabidopsis and rice, only a few ERF genes have been characterized so far. Flower senescence is associated with increased ethylene production in many flowers. However, the characterization of ERF genes in flower senescence has not been reported. In this study, 13 ERF cDNAs were cloned from petunia. Based on the sequence characterization, these PhERFs could be classified into four of the 12 known ERF families. Their predicted amino acid sequences exhibited similarities to ERFs from other plant species. Expression analyses of PhERF mRNAs were performed in corollas and gynoecia of petunia flower. The 13 PhERF genes displayed differential expression patterns and levels during natural flower senescence. Exogenous ethylene accelerates the transcription of the various PhERF genes, and silver thiosulphate (STS) decreased the transcription of several PhERF genes in corollas and gynoecia. PhERF genes of group VII showed a strong association with the rise in ethylene production in both petals and gynoecia, and might be associated particularly with flower senescence in petunia. The effect of sugar, methyl jasmonate, and the plant hormones abscisic acid, salicylic acid, and 6-benzyladenine in regulating the different PhERF transcripts was investigated. Functional nuclear localization signal analyses of two PhERF proteins (PhERF2 and PhERF3) were carried out using fluorescence microscopy. These results supported a role for petunia PhERF genes in transcriptional regulation of petunia flower senescence processes.

Bmp2 Deletion Causes an Amelogenesis Imperfecta Phenotype Via Regulating Enamel Gene Expression

PubMed Central

GUO, FENG; FENG, JUNSHENG; WANG, FENG; LI, WENTONG; GAO, QINGPING; CHEN, ZHUO; SHOFF, LISA; DONLY, KEVIN J.; GLUHAK-HEINRICH, JELICA; CHUN, YONG HEE PATRICIA; HARRIS, STEPHEN E.; MACDOUGALL, MARY; CHEN, SHUO

2015-01-01

Although Bmp2 is essential for tooth formation, the role of Bmp2 during enamel formation remains unknown in vivo. In this study, the role of Bmp2 in regulation of enamel formation was investigated by the Bmp2 conditional knock out (Bmp2 cKO) mice. Teeth of Bmp2 cKO mice displayed severe and profound phenotypes with asymmetric and misshaped incisors as well as abrasion of incisors and molars. Scanning electron microscopy analysis showed that the enamel layer was hypoplastic and enamel lacked a typical prismatic pattern. Teeth from null mice were much more brittle as tested by shear and compressive moduli. Expression of enamel matrix protein genes, amelogenin, enamelin, and enamel-processing proteases, Mmp-20 and Klk4 was reduced in the Bmp2 cKO teeth as reflected in a reduced enamel formation. Exogenous Bmp2 up-regulated those gene expressions in mouse enamel organ epithelial cells. This result for the first time indicates Bmp2 signaling is essential for proper enamel development and mineralization in vivo. PMID:25545831

Bmp2 deletion causes an amelogenesis imperfecta phenotype via regulating enamel gene expression.

PubMed

Guo, Feng; Feng, Junsheng; Wang, Feng; Li, Wentong; Gao, Qingping; Chen, Zhuo; Shoff, Lisa; Donly, Kevin J; Gluhak-Heinrich, Jelica; Chun, Yong Hee Patricia; Harris, Stephen E; MacDougall, Mary; Chen, Shuo

2015-08-01

Although Bmp2 is essential for tooth formation, the role of Bmp2 during enamel formation remains unknown in vivo. In this study, the role of Bmp2 in regulation of enamel formation was investigated by the Bmp2 conditional knock out (Bmp2 cKO) mice. Teeth of Bmp2 cKO mice displayed severe and profound phenotypes with asymmetric and misshaped incisors as well as abrasion of incisors and molars. Scanning electron microscopy analysis showed that the enamel layer was hypoplastic and enamel lacked a typical prismatic pattern. Teeth from null mice were much more brittle as tested by shear and compressive moduli. Expression of enamel matrix protein genes, amelogenin, enamelin, and enamel-processing proteases, Mmp-20 and Klk4 was reduced in the Bmp2 cKO teeth as reflected in a reduced enamel formation. Exogenous Bmp2 up-regulated those gene expressions in mouse enamel organ epithelial cells. This result for the first time indicates Bmp2 signaling is essential for proper enamel development and mineralization in vivo. © 2015 Wiley Periodicals, Inc.

[Genetic transformation of flax (Linum usitatissimum L.) with chimeric GFP-TUA6 gene for visualisation of microtubules].

PubMed

Shisha, E N; Korkhovoĭ, V I; Baer, G Ia; Guzenko, E V; Lemesh, V A; Kartel', N A; Emets, A I; Blium, Ia B

2013-01-01

The data of Agrobacterium-mediated transformation of some Linum usitatissimum cultivars zoned on the territories of Belarus and Ukraine with the plasmid carrying chimeric GFP-TUA6 gene and nptII gene as selectable marker conferring resistance to kanamycin are presented in this study. Transformation was affected by a number of factors including optical density (OD600), time of inoculation of explants with Agrobacterium and co-culture conditions. Transgenic nature of obtained lines was confirmed by PCR analysis. Expression of GFP-TUA6 gene was detected with confocal laser scanning microscopy. The obtained transgenic lines can be used for further functional studies the role of microtubules in the processes of building the flax fibres and resistance to wind.

Osteograft, plastic material for regenerative medicine

NASA Astrophysics Data System (ADS)

Zaidman, A. M.; Korel, A. V.; Shevchenko, A. I.; Shchelkunova, E. I.; Sherman, K. M.; Predein, Yu. A.; Kosareva, O. S.

2016-08-01

Creating tissue-engineering constructs based on the mechanism of cartilage-bone evolution is promising for traumatology and orthopedics. Such a graft was obtained from a chondrograft by transdifferentiation. The hondrograft placed in osteogenic medium is undergoing osteogenic differentiation for 14-30 days. Tissue specificity of the osteograft was studied by morphology, immunohistochemistry, electron microscopy, and the expression of the corresponding genes was estimated. The expression of osteonectin, fibronectin, collagen of type I, izolektin and CD 44 is determined. Alkaline phosphatase and matrix vesicles are determined in osteoblasts. Calcificates are observed in the matrix. Chondrogenic proteins expression is absent. These findings evidence the tissue specificity of the developed osteograft.

The immune response induced by DNA vaccine expressing nfa1 gene against Naegleria fowleri.

PubMed

Kim, Jong-Hyun; Lee, Sang-Hee; Sohn, Hae-Jin; Lee, Jinyoung; Chwae, Yong-Joon; Park, Sun; Kim, Kyongmin; Shin, Ho-Joon

2012-12-01

The pathogenic free-living amoeba, Naegleria fowleri, causes fatal primary amoebic meningoencephalitis in experimental animals and in humans. The nfa1 gene that was cloned from N. fowleri is located on pseudopodia, especially amoebic food cups and plays an important role in the pathogenesis of N. fowleri. In this study, we constructed and characterized retroviral vector and lentiviral vector systems for nfa1 DNA vaccination in mice. We constructed the retroviral vector (pQCXIN) and the lentiviral vector (pCDH) cloned with the egfp-nfa1 gene. The expression of nfa1 gene in Chinese hamster ovary cell and human primary nasal epithelial cell transfected with the pQCXIN/egfp-nfa1 vector or pCDH/egfp-nfa1 vector was observed by fluorescent microscopy and Western blotting analysis. Our viral vector systems effectively delivered the nfa1 gene to the target cells and expressed the Nfa1 protein within the target cells. To evaluate immune responses of nfa1-vaccinated mice, BALB/c mice were intranasally vaccinated with viral particles of each retro- or lentiviral vector expressing nfa1 gene. DNA vaccination using viral vectors expressing nfa1 significantly stimulated the production of Nfa1-specific IgG subclass, as well as IgG levels. In particular, both levels of IgG2a (Th1) and IgG1 (Th2) were significantly increased in mice vaccinated with viral vectors. These results show the nfa1-vaccination induce efficiently Th1 type, as well as Th2 type immune responses. This is the first report to construct viral vector systems and to evaluate immune responses as DNA vaccination in N. fowleri infection. Furthermore, these results suggest that nfal vaccination may be an effective method for treatment of N. fowleri infection.

Transcriptome Analysis of Arbuscular Mycorrhizal Roots during Development of the Prepenetration Apparatus1[W

PubMed Central

Siciliano, Valeria; Genre, Andrea; Balestrini, Raffaella; Cappellazzo, Gilda; deWit, Pierre J.G.M.; Bonfante, Paola

2007-01-01

Information on changes in the plant transcriptome during early interaction with arbuscular mycorrhizal (AM) fungi is still limited since infections are usually not synchronized and plant markers for early stages of colonization are not yet available. A prepenetration apparatus (PPA), organized in epidermal cells during appressorium development, has been reported to be responsible for assembling a trans-cellular tunnel to accommodate the invading fungus. Here, we used PPAs as markers for cell responsiveness to fungal contact to investigate gene expression at this early stage of infection with minimal transcript dilution. PPAs were identified by confocal microscopy in transformed roots of Medicago truncatula expressing green fluorescent protein-HDEL, colonized by the AM fungus Gigaspora margarita. A PPA-targeted suppressive-subtractive cDNA library was built, the cDNAs were cloned and sequenced, and, consequently, 107 putative interaction-specific genes were identified. The expression of a subset of 15 genes, selected by reverse northern dot blot screening, and five additional genes, potentially involved in PPA formation, was analyzed by real-time reverse transcription-polymerase chain reaction and compared with an infection stage, 48 h after the onset of the PPA. Comparison of the expression profile of G. margarita-inoculated wild type and the mycorrhiza-defective dmi3-1 mutant of M. truncatula revealed that an expansin-like gene, expressed in wild-type epidermis during PPA development, can be regarded as an early host marker for successful mycorrhization. A putative Avr9/Cf-9 rapidly elicited gene, found to be up-regulated in the mutant, suggests novel regulatory roles for the DMI3 protein in the early mycorrhization process. PMID:17468219

Effects of Selected Egyptian Honeys on the Cellular Ultrastructure and the Gene Expression Profile of Escherichia coli

PubMed Central

Elkhatib, Walid F.

2016-01-01

The purpose of this study was to: (i) evaluate the antibacterial activities of three Egyptian honeys collected from different floral sources (namely, citrus, clover, and marjoram) against Escherichia coli; (ii) investigate the effects of these honeys on bacterial ultrastructure; and (iii) assess the anti-virulence potential of these honeys, by examining their impacts on the expression of eight selected genes (involved in biofilm formation, quorum sensing, and stress survival) in the test organism. The minimum inhibitory concentration (MIC) of the honey samples against E. coli ATCC 8739 were assessed by the broth microdilution assay in the presence and absence of catalase enzyme. Impacts of the honeys on the cellular ultrastructure and the expression profiles of the selected genes of E. coli were examined using transmission electron microscopy (TEM) and quantitative real-time polymerase chain reaction (qPCR) analysis, respectively. The susceptibility tests showed promising antibacterial activities of all the tested honeys against E. coli. This was supported by the TEM observations, which revealed “ghost” cells lacking DNA, in addition to cells with increased vacuoles, and/or with irregular shrunken cytoplasm. Among the tested honeys, marjoram exhibited the highest total antibacterial activity and the highest levels of peroxide-dependent activity. The qPCR analysis showed that all honey-treated cells share a similar overall pattern of gene expression, with a trend toward reduced expression of the virulence genes of interest. Our results indicate that some varieties of the Egyptian honey have the potential to be effective inhibitor and virulence modulator of E. coli via multiple molecular targets. PMID:26954570

Expression of Olfactory Signaling Genes in the Eye

PubMed Central

Velmeshev, Dmitry; Faghihi, Mohammad; Shestopalov, Valery I.; Slepak, Vladlen Z.

2014-01-01

Purpose To advance our understanding how the outer eye interacts with its environment, we asked which cellular receptors are expressed in the cornea, focusing on G protein-coupled receptors. Methods Total RNA from the mouse cornea was subjected to next-generation sequencing using the Illumina platform. The data was analyzed with TopHat and CuffLinks software packages. Expression of a representative group of genes detected by RNA-seq was further analyzed by RT-PCR and in situ hybridization using RNAscope technology and fluorescent microscopy. Results We generated more than 46 million pair-end reads from mouse corneal RNA. Bioinformatics analysis revealed that the mouse corneal transcriptome reconstructed from these reads represents over 10,000 gene transcripts. We identified 194 GPCR transcripts, of which 96 were putative olfactory receptors. RT-PCR analysis confirmed the presence of several olfactory receptors and related genes, including olfactory marker protein and the G protein associated with olfaction, Gαolf. In situ hybridization showed that mRNA for olfactory marker protein, Gαolf and possibly some olfactory receptors were found in the corneal epithelial cells. In addition to the corneal epithelium, Gαolf was present in the ganglionic and inner nuclear layers of the retina. One of the olfactory receptors, Olfr558, was present primarily in vessels of the eye co-stained with antibodies against alpha-smooth muscle actin, indicating expression in arterioles. Conclusions Several species of mRNA encoding putative olfactory receptors and related genes are expressed in the mouse cornea and other parts of the eye indicating they may play a role in sensing chemicals in the ocular environment. PMID:24789354

Effects of Selected Egyptian Honeys on the Cellular Ultrastructure and the Gene Expression Profile of Escherichia coli.

PubMed

Wasfi, Reham; Elkhatib, Walid F; Khairalla, Ahmed S

2016-01-01

The purpose of this study was to: (i) evaluate the antibacterial activities of three Egyptian honeys collected from different floral sources (namely, citrus, clover, and marjoram) against Escherichia coli; (ii) investigate the effects of these honeys on bacterial ultrastructure; and (iii) assess the anti-virulence potential of these honeys, by examining their impacts on the expression of eight selected genes (involved in biofilm formation, quorum sensing, and stress survival) in the test organism. The minimum inhibitory concentration (MIC) of the honey samples against E. coli ATCC 8739 were assessed by the broth microdilution assay in the presence and absence of catalase enzyme. Impacts of the honeys on the cellular ultrastructure and the expression profiles of the selected genes of E. coli were examined using transmission electron microscopy (TEM) and quantitative real-time polymerase chain reaction (qPCR) analysis, respectively. The susceptibility tests showed promising antibacterial activities of all the tested honeys against E. coli. This was supported by the TEM observations, which revealed "ghost" cells lacking DNA, in addition to cells with increased vacuoles, and/or with irregular shrunken cytoplasm. Among the tested honeys, marjoram exhibited the highest total antibacterial activity and the highest levels of peroxide-dependent activity. The qPCR analysis showed that all honey-treated cells share a similar overall pattern of gene expression, with a trend toward reduced expression of the virulence genes of interest. Our results indicate that some varieties of the Egyptian honey have the potential to be effective inhibitor and virulence modulator of E. coli via multiple molecular targets.

Comparison of cyanobacterial microcystin synthetase (mcy) E gene transcript levels, mcy E gene copies, and biomass as indicators of microcystin risk under laboratory and field conditions.

PubMed

Ngwa, Felexce F; Madramootoo, Chandra A; Jabaji, Suha

2014-08-01

Increased incidences of mixed assemblages of microcystin-producing and nonproducing cyanobacterial strains in freshwater bodies necessitate development of reliable proxies for cyanotoxin risk assessment. Detection of microcystin biosynthetic genes in water blooms of cyanobacteria is generally indicative of the presence of potentially toxic cyanobacterial strains. Although much effort has been devoted toward elucidating the microcystin biosynthesis mechanisms in many cyanobacteria genera, little is known about the impacts of co-occurring cyanobacteria on cellular growth, mcy gene expression, or mcy gene copy distribution. The present study utilized conventional microscopy, qPCR assays, and enzyme-linked immunosorbent assay to study how competition between microcystin-producing Microcystis aeruginosa CPCC 299 and Planktothrix agardhii NIVA-CYA 126 impacts mcyE gene expression, mcyE gene copies, and microcystin concentration under controlled laboratory conditions. Furthermore, analyses of environmental water samples from the Missisquoi Bay, Quebec, enabled us to determine how the various potential toxigenic cyanobacterial biomass proxies correlated with cellular microcystin concentrations in a freshwater lake. Results from our laboratory study indicated significant downregulation of mcyE gene expression in mixed cultures of M. aeruginosa plus P. agardhii on most sampling days in agreement with depressed growth recorded in the mixed cultures, suggesting that interaction between the two species probably resulted in suppressed growth and mcyE gene expression in the mixed cultures. Furthermore, although mcyE gene copies and McyE transcripts were detected in all laboratory and field samples with measureable microcystin levels, only mcyE gene copies showed significant positive correlations (R(2) > 0.7) with microcystin concentrations, while McyE transcript levels did not. These results suggest that mcyE gene copies are better indicators of potential risks from microcystins than McyE transcript levels or conventional biomass proxies, especially in water bodies comprising mixed assemblages of toxic and nontoxic cyanobacteria. © 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Effects of proteasome inhibitor MG-132 on the parasite Schistosoma mansoni

PubMed Central

de Paula, Renato G.; Ornelas, Alice M. M.; Moreira, Érika B. C.; Badoco, Fernanda Rafacho; Magalhães, Lizandra G.; Verjovski-Almeida, Sergio; Rodrigues, Vanderlei

2017-01-01

Proteasome is a proteolytic complex responsible for intracellular protein turnover in eukaryotes, archaea and in some actinobacteria species. Previous work has demonstrated that in Schistosoma mansoni parasites, the proteasome inhibitor MG-132 affects parasite development. However, the molecular targets affected by MG-132 in S. mansoni are not entirely known. Here, we used expression microarrays to measure the genome-wide changes in gene expression of S. mansoni adult worms exposed in vitro to MG-132, followed by in silico functional analyses of the affected genes using Ingenuity Pathway Analysis (IPA). Scanning electron microscopy was used to document changes in the parasites’ tegument. We identified 1,919 genes with a statistically significant (q-value ≤ 0.025) differential expression in parasites treated for 24 h with MG-132, when compared with control. Of these, a total of 1,130 genes were up-regulated and 790 genes were down-regulated. A functional gene interaction network comprised of MG-132 and its target genes, known from the literature to be affected by the compound in humans, was identified here as affected by MG-132. While MG-132 activated the expression of the 26S proteasome genes, it also decreased the expression of 19S chaperones assembly, 20S proteasome maturation, ubiquitin-like NEDD8 and its partner cullin-3 ubiquitin ligase genes. Interestingly, genes that encode proteins related to potassium ion binding, integral membrane component, ATPase and potassium channel activities were significantly down-regulated, whereas genes encoding proteins related to actin binding and microtubule motor activity were significantly up-regulated. MG-132 caused important changes in the worm tegument; peeling, outbreaks and swelling in the tegument tubercles could be observed, which is consistent with interference on the ionic homeostasis in S. mansoni. Finally, we showed the down-regulation of Bax pro-apoptotic gene, as well as up-regulation of two apoptosis inhibitor genes, IAP1 and BRE1, and in contrast, down-regulation of Apaf-1 apoptotic activator, thus suggesting that apoptosis is deregulated in S. mansoni exposed to MG-132. A considerable insight has been gained concerning the potential of MG-132 as a gene expression modulator, and overall the data suggest that the proteasome might be an important molecular target for the design of new drugs against schistosomiasis. PMID:28898250

A shell regeneration assay to identify biomineralization candidate genes in mytilid mussels.

PubMed

Hüning, Anne K; Lange, Skadi M; Ramesh, Kirti; Jacob, Dorrit E; Jackson, Daniel J; Panknin, Ulrike; Gutowska, Magdalena A; Philipp, Eva E R; Rosenstiel, Philip; Lucassen, Magnus; Melzner, Frank

2016-06-01

Biomineralization processes in bivalve molluscs are still poorly understood. Here we provide an analysis of specifically expressed sequences from a mantle transcriptome of the blue mussel, Mytilus edulis. We then developed a novel, integrative shell injury assay to test, whether biomineralization candidate genes highly expressed in marginal and pallial mantle could be induced in central mantle tissue underlying the damaged shell areas. This experimental approach makes it possible to identify gene products that control the chemical micro-environment during calcification as well as organic matrix components. This is unlike existing methodological approaches that work retroactively to characterize calcification relevant molecules and are just able to examine organic matrix components that are present in completed shells. In our assay an orthogonal array of nine 1mm holes was drilled into the left valve, and mussels were suspended in net cages for 20, 29 and 36days to regenerate. Structural observations using stereo-microscopy, SEM and Raman spectroscopy revealed organic sheet synthesis (day 20) as the first step of shell-repair followed by the deposition of calcite crystals (days 20 and 29) and aragonite tablets (day 36). The regeneration period was characterized by time-dependent shifts in gene expression in left central mantle tissue underlying the injured shell, (i) increased expression of two tyrosinase isoforms (TYR3: 29-fold and TYR6: 5-fold) at day 20 with a decline thereafter, (ii) an increase in expression of a gene encoding a nacrein-like protein (max. 100-fold) on day 29. The expression of an acidic Asp-Ser-rich protein was enhanced during the entire regeneration process. This proof-of-principle study demonstrates that genes that are specifically expressed in pallial and marginal mantle tissue can be induced (4 out of 10 genes) in central mantle following experimental injury of the overlying shell. Our findings suggest that regeneration assays can be used systematically to better characterize gene products that are essential for distinct phases of the shell formation process, particularly those that are not incorporated into the organic shell matrix. Copyright © 2016 Elsevier B.V. All rights reserved.

Expression of Foreign Genes Demonstrates the Effectiveness of Pollen-Mediated Transformation in Zea mays.

PubMed

Yang, Liyan; Cui, Guimei; Wang, Yixue; Hao, Yaoshan; Du, Jianzhong; Zhang, Hongmei; Wang, Changbiao; Zhang, Huanhuan; Wu, Shu-Biao; Sun, Yi

2017-01-01

Plant genetic transformation has arguably been the core of plant improvement in recent decades. Efforts have been made to develop in planta transformation systems due to the limitations present in the tissue-culture-based methods. Herein, we report an improved in planta transformation system, and provide the evidence of reporter gene expression in pollen tube, embryos and stable transgenicity of the plants following pollen-mediated plant transformation with optimized sonication treatment of pollen. The results showed that the aeration at 4°C treatment of pollen grains in sucrose prior to sonication significantly improved the pollen viability leading to improved kernel set and transformation efficiency. Scanning electron microscopy observation revealed that the removal of operculum covering pollen pore by ultrasonication might be one of the reasons for the pollen grains to become competent for transformation. Evidences have shown that the eGfp gene was expressed in the pollen tube and embryos, and the Cry1Ac gene was detected in the subsequent T 1 and T 2 progenies, suggesting the successful transfer of the foreign genes to the recipient plants. The Southern blot analysis of Cry1Ac gene in T 2 progenies and PCR-identified Apr gene segregation in T 2 seedlings confirmed the stable inheritance of the transgene. The outcome illustrated that the pollen-mediated genetic transformation system can be widely applied in the plant improvement programs with apparent advantages over tissue-culture-based transformation methods.

Elevated hydrostatic pressure triggers release of OPA1 and cytochrome C, and induces apoptotic cell death in differentiated RGC-5 cells

PubMed Central

Kim, Keun-Young; Lindsey, James D.; Angert, Mila; Patel, Ankur; Scott, Ray T.; Liu, Quan; Crowston, Jonathan G.; Ellisman, Mark H.; Perkins, Guy A.; Weinreb, Robert N.

2009-01-01

Purpose This study was conducted to determine whether elevated hydrostatic pressure alters mitochondrial structure, triggers release of the dynamin-related guanosine triphosphatase (GTPase) optic atrophy type 1 (OPA1) or cytochrome C from mitochondria, alters OPA1 gene expression, and can directly induce apoptotic cell death in cultured retinal ganglion cell (RGC)-5 cells. Methods Differentiated RGC-5 cells were exposed to 30 mmHg for three days in a pressurized incubator. As a control, differentiated RGC-5 cell cultures were incubated simultaneously in a conventional incubator. Live RGC-5 cells were then labeled with MitoTracker Red and mitochondrial morphology was assessed by fluorescence microscopy. Mitochondrial structural changes were also assessed by electron microscopy and three-dimenstional (3D) electron microscope tomography. OPA1 mRNA was measured by Taqman quantitative PCR. The cellular distribution of OPA1 protein and cytochrome C was assessed by immunocytochemistry and western blot. Caspase-3 activation was examined by western blot. Apoptotic cell death was evaluated by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method. Results Mitochondrial fission, characterized by the conversion of tubular fused mitochondria into isolated small organelles, was triggered after three days exposure to elevated hydrostatic pressure. Electron microscopy confirmed the fission and noted no changes to mitochondrial architecture, nor outer membrane rupture. Electron microscope tomography showed that elevated pressure depleted mitochondrial cristae content by fourfold. Elevated hydrostatic pressure increased OPA1 gene expression by 35±14% on day 2, but reduced expression by 36±4% on day 3. Total OPA1 protein content was not changed on day 2 or 3. However, pressure treatment induced release of OPA1 and cytochrome C from mitochondria to the cytoplasm. Elevated pressure also activated caspase-3 and induced apoptotic cell death. Conclusions Elevated hydrostatic pressure triggered mitochondrial changes including mitochondrial fission and abnormal cristae depletion, alteration of OPA1 gene expression, and release of OPA1 and cytochrome C into the cytoplasm before the onset of apoptotic cell death in differentiated RGC-5 cells. These results suggest that sustained moderate pressure elevation may directly damage RGC integrity by injuring mitochondria. PMID:19169378

Wnt signaling is involved in human articular chondrocyte de-differentiation in vitro.

PubMed

Sassi, N; Laadhar, L; Allouche, M; Zandieh-Doulabi, B; Hamdoun, M; Klein-Nulend, J; Makni, S; Sellami, S

2014-01-01

Osteoarthritis is the most prevalent form of arthritis in the world. Certain signaling pathways, such as the wnt pathway, are involved in cartilage pathology. Osteoarthritic chondrocytes undergo morphological and biochemical changes that lead to chondrocyte de-differentiation. We investigated whether the Wnt pathway is involved in de-differentiation of human articular chondrocytes in vitro. Human articular chondrocytes were cultured for four passages in the presence or absence of IL-1 in monolayer or micromass culture. Changes in cell morphology were monitored by light microscopy. Protein and gene expression of chondrocyte markers and Wnt pathway components were determined by Western blotting and qPCR after culture. After culturing for four passages, chondrocytes exhibited a fibroblast-like morphology. Collagen type II and aggrecan protein and gene expression decreased, while collagen type I, matrix metalloproteinase 13, and nitric oxide synthase expressions increased. Wnt molecule expression profiles changed; Wnt5a protein expression, the Wnt target gene, c-jun, and in Wnt pathway regulator, sFRP4 increased. Treatment with IL-1 caused chondrocyte morphology to become more filament-like. This change in morphology was accompanied by extinction of col II expression and increased col I, MMP13 and eNOS expression. Changes in expression of the Wnt pathway components also were observed. Wnt7a decreased significantly, while Wnt5a, LRP5, β-catenin and c-jun expressions increased. Culture of human articular chondrocytes with or without IL-1 not only induced chondrocyte de-differentiation, but also changed the expression profiles of Wnt components, which suggests that the Wnt pathway is involved in chondrocyte de-differentiation in vitro.

Multifunctional nanocarrier based on clay nanotubes for efficient intracellular siRNA delivery and gene silencing.

PubMed

Wu, Hui; Shi, Yinfeng; Huang, Chusen; Zhang, Yang; Wu, Jiahui; Shen, Hebai; Jia, Nengqin

2014-04-01

RNA interference-mediated gene silencing relating to disease has recently emerged as a powerful method in gene therapy. Despite the promises, effective transport of siRNA with minimal side effects remains a challenge. Halloysites are cheap and naturally available aluminosilicate clay nanotubes with high mechanical strength and biocompatibility. In this study, a novel multifunctional nanocarrier based on functionalized halloysite nanotubes (f-HNTs) has been developed via electrostatic layer-by-layer assembling approach for loading and intracellular delivery of therapeutic antisurvivin siRNA and simultaneously tracking their intracellular transport, in which PEI-modified HNTs are used as gene vector, antisurvivin siRNA as gene therapeutic agent, and mercaptoacetic acid-capped CdSe quantum dots as fluorescent labeling probes. The successful assembly of the f-HNTs-siRNA complexes was systematically characterized by transmission electron microscopy (TEM), UV-visible spectrophotometry, Zeta potential measurement, fluorescence spectrophotometry, and electrochemical impedance spectroscopy. Confocal microscopy, biological TEM, and flow cytometry studies revealed that the complexes enabled the efficient intracellular delivery of siRNA for cell-specific gene silencing. MTT assays exhibited that the complexes can enhance antitumor activity. Furthermore, Western blot analysis showed that f-HNTs-mediated siRNA delivery effectively knocked down gene expression of survivin and thereby decreased the levels of target proteins of PANC-1 cells. Therefore, this study suggested that the synthesized f-HNTs were a new effective drug delivery system for potential application in cancer gene therapy.

Detection of Genetically Altered Copper Levels in Drosophila Tissues by Synchrotron X-Ray Fluorescence Microscopy

PubMed Central

Lye, Jessica C.; Hwang, Joab E. C.; Paterson, David; de Jonge, Martin D.; Howard, Daryl L.; Burke, Richard

2011-01-01

Tissue-specific manipulation of known copper transport genes in Drosophila tissues results in phenotypes that are presumably due to an alteration in copper levels in the targeted cells. However direct confirmation of this has to date been technically challenging. Measures of cellular copper content such as expression levels of copper-responsive genes or cuproenzyme activity levels, while useful, are indirect. First-generation copper-sensitive fluorophores show promise but currently lack the sensitivity required to detect subtle changes in copper levels. Moreover such techniques do not provide information regarding other relevant biometals such as zinc or iron. Traditional techniques for measuring elemental composition such as inductively coupled plasma mass spectroscopy are not sensitive enough for use with the small tissue amounts available in Drosophila research. Here we present synchrotron x-ray fluorescence microscopy analysis of two different Drosophila tissues, the larval wing imaginal disc, and sectioned adult fly heads and show that this technique can be used to detect changes in tissue copper levels caused by targeted manipulation of known copper homeostasis genes. PMID:22053217

The effect of culture medium and carrier on explant culture of human limbal epithelium: A comparison of ultrastructure, keratin profile and gene expression.

PubMed

Pathak, Meeta; Olstad, O K; Drolsum, Liv; Moe, Morten C; Smorodinova, Natalia; Kalasova, Sarka; Jirsova, Katerina; Nicolaissen, Bjørn; Noer, Agate

2016-12-01

Patients with limbal stem cell deficiency (LSCD) often experience pain and photophobia due to recurrent epithelial defects and chronic inflammation of the cornea. Successfully restoring a healthy corneal surface in these patients by transplantation of ex vivo expanded human limbal epithelial cells (LECs) may alleviate these symptoms and significantly improve their quality of life. The clinical outcome of transplantation is known to be influenced by the quality of transplanted cells. Presently, several different protocols for cultivation and transplantation of LECs are in use. However, no consensus on an optimal protocol exists. The aim of this study was to examine the effect of culture medium and carrier on the morphology, staining of selected keratins and global gene expression in ex vivo cultured LECs. Limbal biopsies from cadaveric donors were cultured for three weeks on human amniotic membrane (HAM) or on tissue culture coated plastic (PL) in either a complex medium (COM), containing recombinant growth factors, hormones, cholera toxin and fetal bovine serum, or in medium supplemented only with human serum (HS). The expanded LECs were examined by light microscopy (LM), transmission electron microscopy (TEM), immunohistochemistry (IHC) for keratins K3, K7, K8, K12, K13, K14, K15 and K19, as well as microarray and qRT-PCR analysis. The cultured LECs exhibited similar morphology and keratin staining on LM, TEM and IHC examination, regardless of the culture condition. The epithelium was multilayered, with cuboidal basal cells and flattened superficial cells. Cells were attached to each other by desmosomes. Adhesion complexes were observed between basal cells and the underlying carrier in LECs cultured on HAM, but not in LECs cultured on PL. GeneChip Human Gene 2.0 ST microarray (Affymetrix) analysis revealed that 18,653 transcripts were ≥2 fold up or downregulated (p ≤ 0.05). Cells cultured in the same medium (COM or HS) showed more similarities in gene expression than cells cultured on the same carrier (HAM or PL). When each condition was compared to HAM/COM, no statistical difference was found in the transcription level of the selected genes associated with keratin expression, stemness, proliferation, differentiation, apoptosis, corneal wound healing or autophagy. In conclusion, the results indicate that ex vivo cultures of LECs on HAM and PL, using culture media supplemented with COM or HS, yield tissues with similar morphology and keratin staining. The gene expression appears to be more similar in cells cultured in the same medium (COM or HS) compared to cells cultured on the same carrier (HAM or PL). Copyright © 2016 Elsevier Ltd. All rights reserved.

Tumor formation of prostate cancer cells influenced by stromal cells from the transitional or peripheral zones of the normal prostate

PubMed Central

Zhao, Fu-Jun; Han, Bang-Min; Yu, Sheng-Qiang; Xia, Shu-Jie

2009-01-01

This study was designed to investigate the different involvements of prostatic stromal cells from the normal transitional zone (TZ) or peripheral zone (PZ) in the carcinogenesis of prostate cancer (PCa) epithelial cells (PC-3) in vitro and in vivo co-culture models. Ultra-structures and gene expression profiles of primary cultures of human prostatic stromal cells from the normal TZ or PZ were analyzed by electron microscopy and microarray analysis. In vitro and in vivo co-culture models composed of normal TZ or PZ stromal cells and human PCa PC-3 cells were established. We assessed tumor growth and weight in the in vivo nude mice model. There are morphological and ultra-structural differences in stromal cells from TZ and PZ of the normal prostate. In all, 514 differentially expressed genes were selected by microarray analysis; 483 genes were more highly expressed in stromal cells from TZ and 31 were more highly expressed in those from PZ. Co-culture with PZ stromal cells and transforming growth factor-β1 (TGF-β1) increased the tumor growth of PC-3 cells in vitro and in vivo, as well as Bcl-2 expression. On the other hand, stromal cells of TZ suppressed PC-3 cell tumor growth in the mouse model. We conclude that ultra-structures and gene expression differ between the stromal cells from TZ or PZ of the normal prostate, and stroma–epithelium interactions from TZ or PZ might be responsible for the distinct zonal localization of prostate tumor formation. PMID:19122679

Up-regulation of glutathione-related genes, enzyme activities and transport proteins in human cervical cancer cells treated with doxorubicin.

PubMed

Drozd, Ewa; Krzysztoń-Russjan, Jolanta; Marczewska, Jadwiga; Drozd, Janina; Bubko, Irena; Bielak, Magda; Lubelska, Katarzyna; Wiktorska, Katarzyna; Chilmonczyk, Zdzisław; Anuszewska, Elżbieta; Gruber-Bzura, Beata

2016-10-01

Doxorubicin (DOX), one of the most effective anticancer drugs, acts in a variety of ways including DNA damage, enzyme inhibition and generation of reactive oxygen species. Glutathione (GSH) and glutathione-related enzymes including: glutathione peroxidase (GPX), glutathione reductase (GSR) and glutathione S-transferases (GST) may play a role in adaptive detoxification processes in response to the oxidative stress, thus contributing to drug resistance phenotype. In this study, we investigated effects of DOX treatment on expression and activity of GSH-related enzymes and multidrug resistance-associated proteins in cultured human cervical cancer cells displaying different resistance against this drug (HeLa and KB-V1). Determination of expression level of genes encoding GST isoforms and MRP proteins (GCS, GPX, GSR, GSTA1-3, GSTM1, GSTP1, ABCC1-3, MGST1-3) was performed using StellARray™ Technology. Enzymatic activities of GPX and GSR were measured using biochemical methods. Expression of MRP1 was examined by immunofluorescence microscopy. This study showed that native expression levels of GSTM1 and GSTA3 were markedly higher in KB-V1 cells (2000-fold and 200-fold) compared to HeLa cells. Resistant cells have also shown significantly elevated expression of GSTA1 and GSTA2 genes (200-fold and 50-fold) as a result of DOX treatment. In HeLa cells, exposure to DOX increased expression of all genes: GSTM1 (7-fold) and GSTA1-3 (550-fold, 150-fold and 300-fold). Exposure to DOX led to the slight increase of GCS expression as well as GPX activity in KB-V1 cells, while in HeLa cells it did not. Expression of ABCC1 (MRP1) was not increased in any of the tested cell lines. Our results indicate that expression of GSTM1 and GSTA1-3 genes is up-regulated by DOX treatment and suggest that activity of these genes may be associated with drug resistance of the tested cells. At the same time, involvement of MRP1 in DOX resistance in the given experimental conditions is unlikely. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Eye drop delivery of nano-polymeric micelle formulated genes with cornea-specific promoters.

PubMed

Tong, Yaw-Chong; Chang, Shwu-Fen; Liu, Chia-Yang; Kao, Winston W-Y; Huang, Chong Heng; Liaw, Jiahorng

2007-11-01

This study evaluates the eye drop delivery of genes with cornea-specific promoters, i.e., keratin 12 (K12) and keratocan (Kera3.2) promoters, by non-ionic poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) polymeric micelles (PM) to mouse and rabbit eyes, and investigates the underlying mechanisms. Three PM-formulated plasmids (pCMV-Lac Z, pK12-Lac Z and pKera3.2-Lac Z) containing the Lac Z gene for beta-galactosidase (beta-Gal) whose expression was driven by the promoter of either the cytomegalovirus early gene, the keratin 12 gene or the keratocan gene, were characterized by critical micelle concentration (CMC), dynamic light scattering (DLS), and atomic force microscopy (AFM). Transgene expression in ocular tissue after gene delivery was analyzed by 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Gal) color staining, 1,2-dioxetane beta-Gal enzymatic activity measurement, and real-time polymerase chain reaction (PCR) analysis. The delivery mechanisms of plasmid-PM on mouse and rabbit corneas were evaluated by EDTA and RGD (arginine-glycine-aspartic acid) peptide. The sizes of the three plasmid-PM complexes were around 150-200 nm with unimodal distribution. Enhanced stability was found for three plasmid-PM formulations after DNase I treatment. After six doses of eye drop delivery of pK12-Lac Z-PM three times a day, beta-Gal activity was significantly increased in both mouse and rabbit corneas. Stroma-specific Lac Z expression was only found in pKera3.2-Lac Z-PM-treated animals with pretreatment by 5 mM EDTA, an opener of junctions. Lac Z gene expression in both pK12-Lac Z-PM and pKera3.2-Lac Z-PM delivery groups was decreased by RGD peptide pretreatment. Cornea epithelium- and stroma-specific gene expression could be achieved using cornea-specific promoters of keratin 12 and keratocan genes, and the gene was delivered with PM formulation through non-invasive, eye drop in mice and rabbits. The transfection mechanism of plasmid-PM may involve endocytosis and particle size dependent paracellular transport. 2007 John Wiley & Sons, Ltd

Increased expression of activated pSTAT3 and PIM-1 in the pulmonary vasculature of experimental congenital diaphragmatic hernia.

PubMed

Hofmann, Alejandro D; Takahashi, Toshiaki; Duess, Johannes; Gosemann, Jan-Hendrik; Puri, Prem

2015-06-01

Signal transducer and activator of transcription (STAT) protein family (STAT1-6) regulates diverse cellular processes. Recently, the isoform STAT3 has been implicated to play a central role in the pathogenesis of pulmonary hypertension (PH). In human PH activated STAT3 (pSTAT3) was shown to directly trigger expression of the provirus integration site for Moloney murine leukemia virus (Pim-1), which promotes proliferation and resistance to apoptosis in SMCs. We designed this study to investigate the hypothesis that pSTAT3 and Pim-1 pulmonary vascular expression is increased in nitrofen-induced CDH. Pregnant rats were exposed to nitrofen or vehicle on D9.5. Fetuses were sacrificed on D21 and divided into nitrofen (n=16) and control group (n=16). QRT-PCR, western blotting, and confocal-immunofluorescence were performed to determine pulmonary gene and protein expression levels of pSTAT3 and Pim-1. Pulmonary Pim-1 gene expression was significantly increased in the CDH group compared to controls. Western blotting and confocal-microscopy confirmed increased pulmonary protein expression of Pim-1 and increased activation of pSTAT3 in CDH lungs compared to controls. Markedly increased gene and protein expression of Pim-1 and activated pSTAT3 in the pulmonary vasculature of nitrofen-induced CDH lungs suggest that pSTAT3 and Pim-1 are important mediators of PH in nitrofen-induced CDH. Copyright © 2015. Published by Elsevier Inc.

Formulated Delivery of Enzyme/Prodrug and Cytokine Gene Therapy to Promote Immune Reduction of Treated and Remote Tumors in Mouse Models of Prostate Cancer

DTIC Science & Technology

2007-01-01

Cunha GR, Donjacour AA, Matusik RJ, Rosen JM. Prostate cancer in a transgenic mouse . Proc Natl Acad Sci U S A.1995;92(8):3439- 43 . Kanai F...data not shown). GFP expression in all cell lines was confirmed by UV microscopy and flow cytometry . Evaluation of RM1 cells for assessment of CDUPRT...for prostate cancer in a mouse model that imitates the development of human disease. J. Gene Med. (2004) 6(1): 43 -54. 108. MARTINIELLO-WILKS R

Functional Analyses of the Crohn's Disease Risk Gene LACC1.

PubMed

Assadi, Ghazaleh; Vesterlund, Liselotte; Bonfiglio, Ferdinando; Mazzurana, Luca; Cordeddu, Lina; Schepis, Danika; Mjösberg, Jenny; Ruhrmann, Sabrina; Fabbri, Alessia; Vukojevic, Vladana; Percipalle, Piergiorgio; Salomons, Florian A; Laurencikiene, Jurga; Törkvist, Leif; Halfvarson, Jonas; D'Amato, Mauro

2016-01-01

Genetic variation in the Laccase (multicopper oxidoreductase) domain-containing 1 (LACC1) gene has been shown to affect the risk of Crohn's disease, leprosy and, more recently, ulcerative colitis and juvenile idiopathic arthritis. LACC1 function appears to promote fatty-acid oxidation, with concomitant inflammasome activation, reactive oxygen species production, and anti-bacterial responses in macrophages. We sought to contribute to elucidating LACC1 biological function by extensive characterization of its expression in human tissues and cells, and through preliminary analyses of the regulatory mechanisms driving such expression. We implemented Western blot, quantitative real-time PCR, immunofluorescence microscopy, and flow cytometry analyses to investigate fatty acid metabolism-immune nexus (FAMIN; the LACC1 encoded protein) expression in subcellular compartments, cell lines and relevant human tissues. Gene-set enrichment analyses were performed to initially investigate modulatory mechanisms of LACC1 expression. A small-interference RNA knockdown in vitro model system was used to study the effect of FAMIN depletion on peroxisome function. FAMIN expression was detected in macrophage-differentiated THP-1 cells and several human tissues, being highest in neutrophils, monocytes/macrophages, myeloid and plasmacytoid dendritic cells among peripheral blood cells. Subcellular co-localization was exclusively confined to peroxisomes, with some additional positivity for organelle endomembrane structures. LACC1 co-expression signatures were enriched for genes involved in peroxisome proliferator-activated receptors (PPAR) signaling pathways, and PPAR ligands downregulated FAMIN expression in in vitro model systems. FAMIN is a peroxisome-associated protein with primary role(s) in macrophages and other immune cells, where its metabolic functions may be modulated by PPAR signaling events. However, the precise molecular mechanisms through which FAMIN exerts its biological effects in immune cells remain to be elucidated.

[Experimental study of human umbilical cord blood derived stromal cells transfected with recombinant adenoviral vector co-expressing VCAM-1 and GFP].

PubMed

Zhang, Xi; Si, Ying-Jian; Chen, Xing-Hua; Liu, Yao; Gao, Li; Gao, Lei; Peng, Xian-Gui; Wang, Qing-Yu

2008-06-01

This study was aimed to investigate the effect of vcam-1 gene-modified human umbilical cord blood derived stromal cells (CBDSCs) on hematopoietic regulation so as to establish the experimental foundation for further study. The target gene vcam-1 was cloned into the shuttle plasmid with the report gene GFP. The recombinant shuttle plasmid was transformed into BJ5183 bacteria to recombine with backbone vector pAdeasy-l, and the recombinant adenoviral vector ad-vcam-1-gfp was confirmed after transfection with CBDSCs. The results indicated that two fragments of about 9 kb and 2 kb were obtained after digestion of recombinant plasmid pAdTrack-vcam-1 with NotIand XhoI, and single fragment of 600 bp was obtained after amplification with PCR; two fragments of about 31 kb and 4 kb were obtained after digestion of recombinant plasmid pad-vcam-1-gfp with PacI, which suggested a successful homologous recombination. The expression of vcam-1 gene in ad-vcam-1-gfp transfected CBDSCs could be detected by immunocytochemistry, RT-PCR and fluorescent microscopy. It is concluded that the recombinant adenoviral vector ad-vcam-1-gfp has been constructed successfully, and the expression of vcam-1 is up-regulated in CBDSCs transfected by gene ad-vcam-1-gfp.

Phytosynthesis of silver nanoparticles using Artemisia marschalliana Sprengel aerial part extract and assessment of their antioxidant, anticancer, and antibacterial properties

PubMed Central

Salehi, Soheil; Shandiz, Seyed Ataollah Sadat; Ghanbar, Farinaz; Darvish, Mohammad Raouf; Ardestani, Mehdi Shafiee; Mirzaie, Amir; Jafari, Mohsen

2016-01-01

A rapid phytosynthesis of silver nanoparticles (AgNPs) using an extract from the aerial parts of Artemisia marschalliana Sprengel was investigated in this study. The synthesized AgNPs using A. marschalliana extract was analyzed by UV–visible spectroscopy, X-ray diffraction, and Fourier transform infrared spectroscopy and further characterized by transmission electron microscopy, scanning electron microscopy, zeta potential, and energy-dispersive spectroscopy. Characteristic absorption bands of AgNPs were found near 430 nm in the UV–vis spectrum. Energy-dispersive spectroscopy analysis of AgNPs in the energy range 2–4 keV confirmed the silver signal due to surface plasmon resonance. Scanning electron microscopy and transmission electron microscopy results revealed that the AgNPs were mostly spherical with an average size ranging from 5 nm to 50 nm. The zeta potential value of −31 mV confirmed the stability of the AgNPs. AgNPs produced using the aqueous A. marschalliana extract might serve as a potent in vitro antioxidant, as revealed by 2,2-diphenyl-1-picryl hydrazyl assay. The present study demonstrates the anticancer properties of phytosynthesized AgNPs against human gastric carcinoma AGS cells. AgNPs exerted a dose-dependent inhibitory effect on the viability of cells. Real-time polymerase chain reaction was used for the investigation of Bax and Bcl-2 gene expression in cancer and normal cell lines. Our findings show that the mRNA levels of pro-apoptotic Bax gene expression were significantly upregulated, while the expression of anti-apoptotic Bcl-2 was declined in cells treated with AgNPs compared to normal cells. In addition, flow cytometric analysis showed that the number of early and late apoptotic AGS cells was significantly enhanced following treatment with AgNPs as compared to untreated cells. In addition, the AgNPs showed strong antibacterial properties against tested pathogenic bacteria such as Staphylococcus aureus, Bacillus cereus, Acinetobacter baumannii, and Pseudomonas aeruginosa. Based on the obtained data, we suggest that phytosynthesized AgNPs are good alternatives in the treatment of diseases because of the presence of bioactive agents. PMID:27199558

Antibacterial activity and mechanism of action of ε-poly-L-lysine

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

Ye, Ruosong; Xu, Hengyi; Wan, Cuixiang

Highlights: •Antibacterial activity and mechanism of ε-PL against E. coli O157:H7 was investigated. •Critical inhibitory factors toward the growth of E. coli O157:H7 by ε-PL was analyzed. •Cell membrane integrity and cell morphology of E. coli O157:H7 was affected by ε-PL. •A positive correlation between reactive oxygen species levels and ε-PL concentration in E. coli O157:H7 cells. •ε-PL induced the expression of different genes related to oxidative/redox stress, SOS response, virulence. -- Abstract: ε-Poly-L-lysine (ε-PL) is widely used as an antibacterial agent because of its broad antimicrobial spectrum. However, the mechanism of ε-PL against pathogens at the molecular level hasmore » not been elucidated. This study investigated the antibacterial activity and mechanism of ε-PL against Escherichia coli O157:H7 CMCC44828. Propidium monoazide-PCR test results indicated that the threshold condition of ε-PL for complete membrane lysis of E. coli O157:H7 was 10 μg/mL (90% mortality for 5 μg/mL). Further verification of the destructive effect of ε-PL on cell structure was performed by atomic force microscopy and transmission electron microscopy. Results showed a positive correlation between reactive oxygen species (ROS) levels and ε-PL concentration in E. coli O157:H7 cells. Moreover, the mortality of E. coli O157:H7 was reduced when antioxidant N-acetylcysteine was added. Results from real-time quantitative PCR (RT-qPCR) indicated that the expression levels of oxidative stress genes sodA and oxyR were up-regulated 4- and 16-fold, respectively, whereas virulence genes eaeA and espA were down-regulated after ε-PL treatment. Expression of DNA damage response (SOS response) regulon genes recA and lexA were also affected by ε-PL. In conclusion, the antibacterial mechanism of ε-PL against E. coli O157:H7 may be attributed to disturbance on membrane integrity, oxidative stress by ROS, and effects on various gene expressions, such as regulation of oxidative stress, SOS response, and changes in virulence.« less

Repeated and diverse losses of corolla bilateral symmetry in the Lamiaceae.

PubMed

Zhong, Jinshun; Preston, Jill C; Hileman, Lena C; Kellogg, Elizabeth A

2017-05-01

Independent evolution of derived complex characters provides a unique opportunity to assess whether and how similar genetic changes correlate with morphological convergence. Bilaterally symmetrical corollas have evolved multiple times independently from radially symmetrical ancestors and likely represent adaptations to attract specific pollinators. On the other hand, losses of bilateral corolla symmetry have occurred sporadically in various groups, due to either modification of bilaterally symmetrical corollas in late development or early establishment of radial symmetry. This study integrated phylogenetic, scanning electron microscopy (SEM)-based morphological, and gene expression approaches to assess the possible mechanisms underlying independent evolutionary losses of corolla bilateral symmetry. This work compared three species of Lamiaceae having radially symmetrical mature corollas with a representative sister taxon having bilaterally symmetrical corollas and found that each reaches radial symmetry in a different way. Higher core Lamiales share a common duplication in the CYCLOIDEA (CYC ) 2 gene lineage and show conserved and asymmetrical expression of CYC2 clade and RAD genes along the adaxial-abaxial floral axis in species having bilateral corolla symmetry. In Lycopus americanus , the development and expression pattern of La-CYC2A and La-CYC2B are similar to those of their bilaterally symmetrical relatives, whereas the loss of La-RAD expression correlates with a late switch to radial corolla symmetry. In Mentha longifolia , late radial symmetry may be explained by the loss of Ml-CYC2A , and by altered expression of two Ml-CYC2B and Ml-RAD genes . Finally, expanded expression of Cc-CYC2A and Cc-RAD strongly correlates with the early development of radially symmetrical corollas in Callicarpa cathayana . Repeated losses of mature corolla bilateral symmetry in Lamiaceae are not uncommon, and may be achieved by distinct mechanisms and various changes to symmetry genes, including the loss of a CYC2 clade gene from the genome, and/or contraction, expansion or alteration of CYC2 clade and RAD -like gene expression. © The Author 2017. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Real-time imaging of Huntingtin aggregates diverting target search and gene transcription

PubMed Central

Li, Li; Liu, Hui; Dong, Peng; Li, Dong; Legant, Wesley R; Grimm, Jonathan B; Lavis, Luke D; Betzig, Eric; Tjian, Robert; Liu, Zhe

2016-01-01

The presumptive altered dynamics of transient molecular interactions in vivo contributing to neurodegenerative diseases have remained elusive. Here, using single-molecule localization microscopy, we show that disease-inducing Huntingtin (mHtt) protein fragments display three distinct dynamic states in living cells – 1) fast diffusion, 2) dynamic clustering and 3) stable aggregation. Large, stable aggregates of mHtt exclude chromatin and form 'sticky' decoy traps that impede target search processes of key regulators involved in neurological disorders. Functional domain mapping based on super-resolution imaging reveals an unexpected role of aromatic amino acids in promoting protein-mHtt aggregate interactions. Genome-wide expression analysis and numerical simulation experiments suggest mHtt aggregates reduce transcription factor target site sampling frequency and impair critical gene expression programs in striatal neurons. Together, our results provide insights into how mHtt dynamically forms aggregates and disrupts the finely-balanced gene control mechanisms in neuronal cells. DOI: http://dx.doi.org/10.7554/eLife.17056.001 PMID:27484239

Osteograft, plastic material for regenerative medicine

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

Zaidman, A. M., E-mail: AZaydman@niito.ru; Korel, A. V., E-mail: AKorel@niito.ru; Shchelkunova, E. I., E-mail: EShelkunova@niito.ru

Creating tissue-engineering constructs based on the mechanism of cartilage-bone evolution is promising for traumatology and orthopedics. Such a graft was obtained from a chondrograft by transdifferentiation. The hondrograft placed in osteogenic medium is undergoing osteogenic differentiation for 14–30 days. Tissue specificity of the osteograft was studied by morphology, immunohistochemistry, electron microscopy, and the expression of the corresponding genes was estimated. The expression of osteonectin, fibronectin, collagen of type I, izolektin and CD 44 is determined. Alkaline phosphatase and matrix vesicles are determined in osteoblasts. Calcificates are observed in the matrix. Chondrogenic proteins expression is absent. These findings evidence the tissuemore » specificity of the developed osteograft.« less

An RNA-seq transcriptome analysis of floral buds of an interspecific Brassica hybrid between B. carinata and B. napus.

PubMed

Chu, Pu; Liu, Huijuan; Yang, Qing; Wang, Yankun; Yan, Guixia; Guan, Rongzhan

2014-12-01

Interspecific hybridizations promote gene transfer between species and play an important role in plant speciation and crop improvement. However, hybrid sterility that commonly found in the first generation of hybrids hinders the utilization of interspecific hybridization. The combination of divergent parental genomes can create extensive transcriptome variations, and to determine these gene expression alterations and their effects on hybrids, an interspecific Brassica hybrid of B. carinata × B. napus was generated. Scanning electron microscopy analysis indicated that some of the hybrid pollen grains were irregular in shape and exhibited abnormal exine patterns compared with those from the parents. Using the Illumina HiSeq 2000 platform, 39,598, 32,403 and 42,208 genes were identified in flower buds of B. carinata cv. W29, B. napus cv. Zhongshuang 11 and their hybrids, respectively. The differentially expressed genes were significantly enriched in pollen wall assembly, pollen exine formation, pollen development, pollen tube growth, pollination, gene transcription, macromolecule methylation and translation, which might be associated with impaired fertility in the F1 hybrid. These results will shed light on the mechanisms underlying the low fertility of the interspecific hybrids and expand our knowledge of interspecific hybridization.

The transcription factor mohawk homeobox regulates homeostasis of the periodontal ligament.

PubMed

Koda, Naoki; Sato, Tempei; Shinohara, Masahiro; Ichinose, Shizuko; Ito, Yoshiaki; Nakamichi, Ryo; Kayama, Tomohiro; Kataoka, Kensuke; Suzuki, Hidetsugu; Moriyama, Keiji; Asahara, Hiroshi

2017-01-15

The periodontal ligament (PDL), which connects the teeth to the alveolar bone, is essential for periodontal tissue homeostasis. Although the significance of the PDL is recognized, molecular mechanisms underlying PDL function are not well known. We report that mohawk homeobox (Mkx), a tendon-specific transcription factor, regulates PDL homeostasis by preventing its degeneration. Mkx is expressed in the mouse PDL at the age of 10 weeks and expression remained at similar levels at 12 months. In Mkx -/- mice, age-dependent expansion of the PDL at the maxillary first molar (M1) furcation area was observed. Transmission electron microscopy (TEM) revealed that Mkx -/- mice presented collagen fibril degeneration in PDL with age, while the collagen fibril diameter gradually increased in Mkx +/+ mice. PDL cells lost their shape in Mkx -/- mice, suggesting changes in PDL properties. Microarray and quantitative polymerase chain reaction (qPCR) analyses of Mkx -/- PDL revealed an increase in osteogenic gene expression and no change in PDL- and inflammatory-related gene expression. Additionally, COL1A1 and COL1A2 were upregulated in Mkx-overexpressing human PDL fibroblasts, whereas osteogenic genes were downregulated. Our results indicate that Mkx prevents PDL degeneration by regulating osteogenesis. © 2017. Published by The Company of Biologists Ltd.

Temporal and spatial regulation of mRNA export: Single particle RNA-imaging provides new tools and insights

PubMed Central

Heinrich, Stephanie; Derrer, Carina Patrizia; Lari, Azra; Weis, Karsten; Montpetit, Ben

2017-01-01

The transport of messenger RNAs (mRNAs) from the nucleus to cytoplasm is an essential step in the gene expression program of all eukaryotes. Recent technological advances in the areas of RNA-labeling, microscopy, and sequencing are leading to novel insights about mRNA biogenesis and export. This includes quantitative single molecule imaging (SMI) of RNA molecules in live cells, which is providing knowledge of the spatial and temporal dynamics of the export process. As this information becomes available, it leads to new questions, the reinterpretation of previous findings, and revised models of mRNA export. In this review, we will briefly highlight some of these recent findings and discuss how live cell SMI approaches may be used to further our current understanding of mRNA export and gene expression. PMID:28052353

Effects of vitrification on nuclear maturation, ultrastructural changes and gene expression of canine oocytes

PubMed Central

2010-01-01

Background Cryopreservation of oocytes, which is an interesting procedure to conserve female gametes, is an essential part of reproductive biotechnology. The objective of the present study was to investigate the effects of vitrification on nuclear maturation, ultrastructural changes and gene expression of canine oocytes. Methods Immature oocytes (germinal vesicles) isolated from ovaries of normal bitches (> 6 months of age) were either vitrified in open pulled straw (OPS) using 20% ethylene glycol (EG) and 20% dimethyl sulfoxide (DMSO) as vitrification solution or exposed to vitrification solution without subjected to liquid nitrogen. After warming, oocytes were investigated for nuclear maturation following in vitro maturation (IVM), ultrastructural changes using transmission electron microscopy (TEM) and gene expression using RT-PCR. Fresh immature oocytes were used as the control group. Results The rate of resumption of meiosis in vitrified-warmed oocytes (53.4%) was significantly (P < 0.05) lower than those of control (93.8%) and exposure (91.4%) groups. However, there were no statistically significant differences among groups in the rates of GV oocytes reaching the maturation stage (metaphase II, MII). The ultrastructural alterations revealed by TEM showed that cortical granules, mitochondria, lipid droplets and smooth endoplasmic reticulum (SER) were affected by vitrification procedures. RT-PCR analysis for gene expression revealed no differences in HSP70, Dnmt1, SOD1 and BAX genes among groups, whereas Bcl2 was strongly expressed in vitrified-warmed group when compared to the control. Conclusion Immature canine oocytes were successfully cryopreserved, resumed meiosis and developed to the MII stage. The information obtained in this study is crucial for the development of an effective method to cryopreserve canine oocytes for establishment of genetic banks of endangered canid species. PMID:20565987

Inhibition of MMP-2 gene expression with small interfering RNA in rabbit vascular smooth muscle cells.

PubMed

Hlawaty, Hanna; San Juan, Aurélie; Jacob, Marie-Paule; Vranckx, Roger; Letourneur, Didier; Feldman, Laurent J

2007-12-01

Matrix metalloproteinase-2 (MMP-2) is constitutively expressed in vascular smooth muscle cells (VSMCs). Using small interfering RNA (siRNA), we evaluated the effect of MMP-2 inhibition in VSMCs in vitro and ex vivo. Rabbit VSMCs were transfected in vitro with 50 nmol/l MMP-2 siRNA or scramble siRNA. Flow cytometry and confocal microscopy showed cellular uptake of siRNA in approximately 80% of VSMCs. MMP-2 mRNA levels evaluated by real-time RT-PCR, pro-MMP-2 activity from conditioned culture media evaluated by gelatin zymography, and VSMC migration were reduced by 44 +/- 19%, 43 +/- 14%, and 36 +/- 14%, respectively, in MMP-2 siRNA-transfected compared with scramble siRNA-transfected VSMCs (P < 0.005 for all). Ex vivo MMP-2 siRNA transfection was performed 2 wk after balloon injury of hypercholesterolemic rabbit carotid arteries. Fluorescence microscopy showed circumferential siRNA uptake in neointimal cells. Gelatin zymography of carotid artery culture medium demonstrated a significant decrease of pro-MMP-2 activity in MMP-2 siRNA-transfected compared with scramble siRNA-transfected arteries (P < 0.01). Overall, our results demonstrate that in vitro MMP-2 siRNA transfection in VSMCs markedly inhibits MMP-2 gene expression and VSMC migration and that ex vivo delivery of MMP-2 siRNA in balloon-injured arteries reduces pro-MMP-2 activity in neointimal cells, suggesting that siRNA could be used to modify arterial biology in vivo.

Preparation and evaluation of MS2 bacteriophage-like particles packaging hepatitis E virus RNA.

PubMed

Wang, Shen; Liu, Ying; Li, Dandan; Zhou, Tiezhong; Gao, Shenyang; Zha, Enhui; Yue, Xiqing

2016-10-01

Hepatitis E virus (HEV) is the pathogen causing hepatitis E (HE). It arouses global public health concern since it is a zoonotic disease. The objective of this letter is to report a cost-effective internal control prepared for monitoring procedures of HEV reverse transcriptase (RT)-PCR detection. A selected conserved HEV RNA fragment was integrated into the downstream of the truncated MS2 bacteriophage genome based on Armored RNA technology. The resulting MS2-HEV gene harbored by the pET-28b-MS2-HEV plasmid was transformed into E. coli BL21(DE3) for expression analysis by SDS-PAGE. The expression products were purified and concentrated by ultrasonication and ultrafiltration separation. The morphology and stability properties of the virus-like particles (VLPs) were evaluated by electron microscopy scanning and nuclease challenges, respectively. SDS-PAGE results showed that the constructed MS2-HEV gene expressed efficiently and the purity of the VLPs was highly consistent with the result in electron microscopy. Stability evaluation results demonstrated that the prepared VLPs exhibited strong resistance to DNase I and RNase A attacks and also performed long-lasting protection of coated HEV RNA for at least 4 months at -20°C. These data revealed that the prepared VLPs meet the basic requirements of use as internal control material in the HEV RNA amplification assay. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Expression Profiles of TGF-β and TLR Pathways in Porphyromonas gingivalis and Prevotella intermedia Challenged Osteoblasts

PubMed Central

Aydin, Kubra; Ekinci, Fatma Yesim; Korachi, May

2015-01-01

Background: The presence of certain oral pathogens at implant sites can hinder the osseointegration process. However, it is unclear how and by what microorganisms it happens. Objectives: This study investigated whether the presence of oral pathogens of Porphyromonas gingivalis and Prevotella intermedia individually, play a role in the failure of bone formation by determining the expression profiles of Transforming Growth Factor Beta (TGF-β/Bone Morphogenic Protein (BMP) and Toll-Like Receptor (TLR) pathways in challenged osteoblasts. Materials and Methods: Cell viability of P. gingivalis and P. intermedia challenged osteoblasts were determined by WST assay. Changes in osteoblast morphology and inhibition of mineralization were observed by Scanning Electron Microscopy (SEM) and Von Kossa staining, respectively. Expression of TGF-β and TLR pathway genes on challenged cells were identified by RT profiler array. Both P. gingivalis and P. intermedia challenges resulted in reduced viability and mineralization of osteoblasts. Results: Viability was reduced to 56.8% (P. gingivalis) and 52.75% (P. intermedia) at 1000 multiplicity. Amongst 48 genes examined, expressions of BMPER, SMAD1, IL8 and NFRKB were found to be highly upregulated by both bacterial challenges (Fold Change > 4). Conclusions: P. gingivalis and P. intermedia could play a role in implant failure by changing the expression profiles of genes related to bone formation and resorption. PMID:26034550

Molecular and immunohistochemical analyses of cardiac troponin T during cardiac development in the Mexican axolotl, Ambystoma mexicanum.

PubMed

Zhang, C; Pietras, K M; Sferrazza, G F; Jia, P; Athauda, G; Rueda-de-Leon, E; Rveda-de-Leon, E; Maier, J A; Dube, D K; Lemanski, S L; Lemanski, L F

2007-01-01

The Mexican axolotl, Ambystoma mexicanum, is an excellent animal model for studying heart development because it carries a naturally occurring recessive genetic mutation, designated gene c, for cardiac nonfunction. The double recessive mutants (c/c) fail to form organized myofibrils in the cardiac myoblasts resulting in hearts that fail to beat. Tropomyosin expression patterns have been studied in detail and show dramatically decreased expression in the hearts of homozygous mutant embryos. Because of the direct interaction between tropomyosin and troponin T (TnT), and the crucial functions of TnT in the regulation of striated muscle contraction, we have expanded our studies on this animal model to characterize the expression of the TnT gene in cardiac muscle throughout normal axolotl development as well as in mutant axolotls. In addition, we have succeeded in cloning the full-length cardiac troponin T (cTnT) cDNA from axolotl hearts. Confocal microscopy has shown a substantial, but reduced, expression of TnT protein in the mutant hearts when compared to normal during embryonic development. 2006 Wiley-Liss, Inc.

Beyond the known functions of the CCR4-NOT complex in gene expression regulatory mechanisms: New structural insights to unravel CCR4-NOT mRNA processing machinery.

PubMed

Ukleja, Marta; Valpuesta, José María; Dziembowski, Andrzej; Cuellar, Jorge

2016-10-01

Large protein assemblies are usually the effectors of major cellular processes. The intricate cell homeostasis network is divided into numerous interconnected pathways, each controlled by a set of protein machines. One of these master regulators is the CCR4-NOT complex, which ultimately controls protein expression levels. This multisubunit complex assembles around a scaffold platform, which enables a wide variety of well-studied functions from mRNA synthesis to transcript decay, as well as other tasks still being identified. Solving the structure of the entire CCR4-NOT complex will help to define the distribution of its functions. The recently published three-dimensional reconstruction of the complex, in combination with the known crystal structures of some of the components, has begun to address this. Methodological improvements in structural biology, especially in cryoelectron microscopy, encourage further structural and protein-protein interaction studies, which will advance our comprehension of the gene expression machinery. © 2016 WILEY Periodicals, Inc.

An Endogenous Accelerator for Viral Gene Expression Confers a Fitness Advantage

PubMed Central

Teng, Melissa W.; Bolovan-Fritts, Cynthia; Dar, Roy D.; Womack, Andrew; Simpson, Michael L.; Shenk, Thomas; Weinberger, Leor S.

2012-01-01

Many signaling circuits face a fundamental tradeoff between accelerating their response speed while maintaining final levels below a cytotoxic threshold. Here, we describe a transcriptional circuitry that dynamically converts signaling inputs into faster rates without amplifying final equilibrium levels. Using time-lapse microscopy, we find that transcriptional activators accelerate human cytomegalovirus (CMV) gene expression in single cells without amplifying steady-state expression levels, and this acceleration generates a significant replication advantage. We map the accelerator to a highly self-cooperative transcriptional negative-feedback loop (Hill coefficient ~ 7) generated by homo-multimerization of the virus’s essential transactivator protein IE2 at nuclear PML bodies. Eliminating the IE2-accelerator circuit reduces transcriptional strength through mislocalization of incoming viral genomes away from PML bodies and carries a heavy fitness cost. In general, accelerators may provide a mechanism for signal-transduction circuits to respond quickly to external signals without increasing steady-state levels of potentially cytotoxic molecules. PMID:23260143

Induction of apoptosis in HeLa cancer cells by an ultrasonic-mediated synthesis of curcumin-loaded chitosan-alginate-STPP nanoparticles.

PubMed

Ahmadi, Fatemeh; Ghasemi-Kasman, Maryam; Ghasemi, Shahram; Gholamitabar Tabari, Maryam; Pourbagher, Roghayeh; Kazemi, Sohrab; Alinejad-Mir, Ali

2017-01-01

Natural herbal compounds have been widely introduced as an alternative therapeutic approach in cancer therapy. Despite potent anticancer activity of curcumin, its clinical application has been limited because of low water solubility and resulting poor bioavailability. In this study, we designed a novel ultrasonic-assisted method for the synthesis of curcumin-loaded chitosan-alginate-sodium tripolyphosphate nanoparticles (CS-ALG-STPP NPs). Furthermore, antitumor effect of curcumin-loaded NPs was evaluated in vitro. Field emission scanning electron microscopy (FE-SEM) and atomic force microscopy (AFM) were used to characterize the properties of NPs. Antitumor activity of curcumin-loaded NPs was assessed by using MTT and quantitative real-time polymerase chain reaction (qRT-PCR). FE-SEM and AFM data revealed the spherical morphology, and the average size of NPs was <50 nm. In vitro cytotoxicity assay suggested that curcumin-loaded CS-ALG-STPP NPs displayed significant antitumor activity compared with the free curcumin. Gene expression level analyses showed that curcumin NPs significantly increased the apoptotic gene expression. Collectively, our results suggest that curcumin-loaded NPs significantly suppressed proliferation and promoted the induction of apoptosis in human cervical epithelioid carcinoma cancer cells, which might be regarded as an effective alternative strategy for cancer therapy.

Induction of apoptosis in HeLa cancer cells by an ultrasonic-mediated synthesis of curcumin-loaded chitosan–alginate–STPP nanoparticles

PubMed Central

Ahmadi, Fatemeh; Ghasemi-Kasman, Maryam; Ghasemi, Shahram; Gholamitabar Tabari, Maryam; Pourbagher, Roghayeh; Kazemi, Sohrab; Alinejad-Mir, Ali

2017-01-01

Natural herbal compounds have been widely introduced as an alternative therapeutic approach in cancer therapy. Despite potent anticancer activity of curcumin, its clinical application has been limited because of low water solubility and resulting poor bioavailability. In this study, we designed a novel ultrasonic-assisted method for the synthesis of curcumin-loaded chitosan–alginate–sodium tripolyphosphate nanoparticles (CS-ALG-STPP NPs). Furthermore, antitumor effect of curcumin-loaded NPs was evaluated in vitro. Field emission scanning electron microscopy (FE-SEM) and atomic force microscopy (AFM) were used to characterize the properties of NPs. Antitumor activity of curcumin-loaded NPs was assessed by using MTT and quantitative real-time polymerase chain reaction (qRT-PCR). FE-SEM and AFM data revealed the spherical morphology, and the average size of NPs was <50 nm. In vitro cytotoxicity assay suggested that curcumin-loaded CS-ALG-STPP NPs displayed significant antitumor activity compared with the free curcumin. Gene expression level analyses showed that curcumin NPs significantly increased the apoptotic gene expression. Collectively, our results suggest that curcumin-loaded NPs significantly suppressed proliferation and promoted the induction of apoptosis in human cervical epithelioid carcinoma cancer cells, which might be regarded as an effective alternative strategy for cancer therapy. PMID:29238191

Histophilus somni biofilm formation in cardiopulmonary tissue of the bovine host following respiratory challenge.

PubMed

Sandal, Indra; Shao, Jian Q; Annadata, Satish; Apicella, Michael A; Boye, Mette; Jensen, Tim K; Saunders, Geoffrey K; Inzana, Thomas J

2009-02-01

Biofilms form in a variety of host sites following infection with many bacterial species. However, the study of biofilms in a host is hindered due to the lack of protocols for the proper experimental investigation of biofilms in vivo. Histophilus somni is an agent of respiratory and systemic diseases in bovines, and readily forms biofilms in vitro. In the present study the capability of H. somni to form biofilms in cardiopulmonary tissue following experimental respiratory infection in the bovine host was examined by light microscopy, transmission electron microscopy, immunoelectron microscopy of ultrathin cryosections, scanning electron microscopy of freeze-fractured samples, and fluorescent in situ hybridization. Biofilms were evident and most prominent in the myocardium, and were associated with a large amount of amorphous extracellular material. Furthermore, Pasteurella multocida was often cultured with H. somni from heart and lung samples. Transposon mutagenesis of H. somni strain 2336 resulted in the generation of mutants that expressed more or less biofilm than the parent strain. Six mutants deficient in biofilm formation had an insertion in the gene encoding for a homolog of filamentous haemagglutinin (FHA), predicted to be involved in attachment. Thus, this investigation demonstrated that H. somni is capable of forming a biofilm in its natural host, that such a biofilm may be capable of harboring other bovine respiratory disease pathogens, and that the genes responsible for biofilm formation can be identified by transposon mutagenesis.

The effect of titanium dioxide nanoparticles on antioxidant gene expression in tilapia ( Oreochromis niloticus)

NASA Astrophysics Data System (ADS)

Varela-Valencia, Ruth; Gómez-Ortiz, Nikte; Oskam, Gerko; de Coss, Romeo; Rubio-Piña, Jorge; del Río-García, Marcela; Albores-Medina, Arnulfo; Zapata-Perez, Omar

2014-04-01

The reactivity of nanoparticles (NPs) in biological systems is well recognized, but there are huge gaps in our understanding of NP toxicity in fish, despite a number of recent ecotoxicity studies. Therefore, the aim of this research was to evaluate the effect of titanium dioxide NPs (TiO2-NPs) on antioxidant gene expression in the tilapia, Oreochromis niloticus. First, different sizes, shapes, and phases of TiO2-NPs were synthesized and characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), and dynamic light scattering (DLS). Fish were injected intraperitoneally with different concentrations (0.1, 1.0, 10.0 mg/L), sizes (7, 14, and 21 nm), and phases (anatase and rutile) of TiO2-NPs, and sacrificed 3, 6, 12, and 24 h after injection, when their livers were removed. Total RNA was extracted, and expression of the catalase ( CAT), glutathione- S-transferase ( GST), and superoxide dismutase ( SOD) genes was assessed by real-time polymerase chain reaction (RT-PCR). The results showed that injection of 1.0 mg/L TiO2-NPs induced an initial mild increase in CAT, GST, and SOD gene expression in tilapia, after which transcript levels decreased. Fish injected with 7 and 14 nm TiO2-NPs showed an increase in antioxidant transcript levels 6 h after treatment. Finally, the rutile form generated stronger induction of the GST gene than anatase TiO2-NPs during the first 6 h after injection, which suggests that exposure to rutile causes higher levels of reactive oxygen species to be produced.

Involvements of PCD and changes in gene expression profile during self-pruning of spring shoots in sweet orange (Citrus sinensis).

PubMed

Zhang, Jin-Zhi; Zhao, Kun; Ai, Xiao-Yan; Hu, Chun-Gen

2014-10-13

Citrus shoot tips abscise at an anatomically distinct abscission zone (AZ) that separates the top part of the shoots into basal and apical portions (citrus self-pruning). Cell separation occurs only at the AZ, which suggests its cells have distinctive molecular regulation. Although several studies have looked into the morphological aspects of self-pruning process, the underlying molecular mechanisms remain unknown. In this study, the hallmarks of programmed cell death (PCD) were identified by TUNEL experiments, transmission electron microscopy (TEM) and histochemical staining for reactive oxygen species (ROS) during self-pruning of the spring shoots in sweet orange. Our results indicated that PCD occurred systematically and progressively and may play an important role in the control of self-pruning of citrus. Microarray analysis was used to examine transcriptome changes at three stages of self-pruning, and 1,378 differentially expressed genes were identified. Some genes were related to PCD, while others were associated with cell wall biosynthesis or metabolism. These results strongly suggest that abscission layers activate both catabolic and anabolic wall modification pathways during the self-pruning process. In addition, a strong correlation was observed between self-pruning and the expression of hormone-related genes. Self-pruning plays an important role in citrus floral bud initiation. Therefore, several key flowering homologs of Arabidopsis and tomato shoot apical meristem (SAM) activity genes were investigated in sweet orange by real-time PCR and in situ hybridization, and the results indicated that these genes were preferentially expressed in SAM as well as axillary meristem. Based on these findings, a model for sweet orange spring shoot self-pruning is proposed, which will enable us to better understand the mechanism of self-pruning and abscission.

Myozenin: An α-actinin- and γ-filamin-binding protein of skeletal muscle Z lines

PubMed Central

Takada, Fumio; Woude, Douglas L. Vander; Tong, Hui-Qi; Thompson, Terri G.; Watkins, Simon C.; Kunkel, Louis M.; Beggs, Alan H.

2001-01-01

To better understand the structure and function of Z lines, we used sarcomeric isoforms of α-actinin and γ-filamin to screen a human skeletal muscle cDNA library for interacting proteins by using the yeast two-hybrid system. Here we describe myozenin (MYOZ), an α-actinin- and γ-filamin-binding Z line protein expressed predominantly in skeletal muscle. Myozenin is predicted to be a 32-kDa, globular protein with a central glycine-rich domain flanked by α-helical regions with no strong homologies to any known genes. The MYOZ gene has six exons and maps to human chromosome 10q22.1-q22.2. Northern blot analysis demonstrated that this transcript is expressed primarily in skeletal muscle with significantly lower levels of expression in several other tissues. Antimyozenin antisera stain skeletal muscle in a sarcomeric pattern indistinguishable from that seen by using antibodies for α-actinin, and immunogold electron microscopy confirms localization specifically to Z lines. Thus, myozenin is a skeletal muscle Z line protein that may be a good candidate gene for limb-girdle muscular dystrophy or other neuromuscular disorders. PMID:11171996

Comparison of GeneXpert MTB/RIF assay and LED-FM microscopy for the diagnosis of extra pulmonary tuberculosis in Khyber Pakhtunkhwa, Pakistan.

PubMed

Khan, Anwar Sheed; Ali, Sajid; Khan, Muhammad Tahir; Ahmed, Sajjad; Khattak, Yasir; Abduljabbar; Irfan, Muhammad; Sajjad, Wasim

2018-04-27

GeneXpert is one of the recent technological instruments used to diagnose tuberculosis in a short span of time. In this study, the performance of GeneXpert MTB/RIF assay for the diagnosis of extrapulmonary tuberculosis (EPTB) was compared with light-emitting diode Fluorescent Microscopy (LED-FM) in Khyber Pakhtunkhwa, Pakistan. A total of 737 EPTB samples were collected from tuberculosis (TB) suspected patients. Out of these samples, male to female ratio was 53% (n=390) to 47% (n=347) respectively. The sensitivity and specificity was 73% and 100% for GeneXpert, while 40% and 100% for LED-FM microscopy. This shows that the sensitivity of GeneXpert is 40-50%, higher than LED-FM microscopy. GeneXpert also detected low number of bacilli as compared to LED-FM microscopy. Copyright © 2018 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Genetic control of ColE1 plasmid stability that is independent of plasmid copy number regulation.

PubMed

Standley, Melissa S; Million-Weaver, Samuel; Alexander, David L; Hu, Shuai; Camps, Manel

2018-06-16

ColE1-like plasmid vectors are widely used for expression of recombinant genes in E. coli. For these vectors, segregation of individual plasmids into daughter cells during cell division appears to be random, making them susceptible to loss over time when no mechanisms ensuring their maintenance are present. Here we use the plasmid pGFPuv in a recA relA strain as a sensitized model to study factors affecting plasmid stability in the context of recombinant gene expression. We find that in this model, plasmid stability can be restored by two types of genetic modifications to the plasmid origin of replication (ori) sequence: point mutations and a novel 269 nt duplication at the 5' end of the plasmid ori, which we named DAS (duplicated anti-sense) ori. Combinations of these modifications produce a range of copy numbers and of levels of recombinant expression. In direct contradiction with the classic random distribution model, we find no correlation between increased plasmid copy number and increased plasmid stability. Increased stability cannot be explained by reduced levels of recombinant gene expression either. Our observations would be more compatible with a hybrid clustered and free-distribution model, which has been recently proposed based on detection of individual plasmids in vivo using super-resolution fluorescence microscopy. This work suggests a role for the plasmid ori in the control of segregation of ColE1 plasmids that is distinct from replication initiation, opening the door for the genetic regulation of plasmid stability as a strategy aimed at enhancing large-scale recombinant gene expression or bioremediation.

Novel strategies to enforce an epithelial phenotype in mesenchymal cells

PubMed Central

Dragoi, Ana-Maria; Swiss, Rachel; Gao, Beile; Agaisse, Hervé

2014-01-01

E-cadherin downregulation in cancer cells is associated with epithelial-to-mesenchymal transition (EMT) and metastatic prowess, but the underlying mechanisms are incompletely characterized. In this study, we probed E-cadherin expression at the plasma membrane as a functional assay to identify genes involved in E-cadherin downregulation. The assay was based on the E-cadherin-dependent invasion properties of the intracellular pathogen Listeria monocytogenes. On the basis of a functional readout, automated microscopy and computer-assisted image analysis were used to screen siRNAs targeting 7,000 human genes. The validity of the screen was supported by its definion of several known regulators of E-cadherin expression, including ZEB1, HDAC1 and MMP14. We identified three new regulators (FLASH, CASP7 and PCGF1), the silencing of which was sufficient to restore high levels of E-cadherin transcription. Additionally, we identified two new regulators (FBXL5 and CAV2), the silencing of which was sufficient to increase E-cadherin expression at a post-transcriptional level. FLASH silencing regulated the expression of E-cadherin and other ZEB1-dependent genes, through post-transcriptional regulation of ZEB1, but it also regulated the expression of numerous ZEB1-independent genes with functions predicted to contribute to a restoration of the epithelial phenotype. Finally, we also report the identification of siRNA duplexes that potently restored the epithelial phenotype by mimicking the activity of known and putative microRNAs. Our findings suggest new ways to enforce epithelial phenotypes as a general strategy to treat cancer by blocking invasive and metastatic phenotypes associated with EMT. PMID:24845104

Generation of Mast Cells from Mouse Fetus: Analysis of Differentiation and Functionality, and Transcriptome Profiling Using Next Generation Sequencer

PubMed Central

Fukuishi, Nobuyuki; Igawa, Yuusuke; Kunimi, Tomoyo; Hamano, Hirofumi; Toyota, Masao; Takahashi, Hironobu; Kenmoku, Hiromichi; Yagi, Yasuyuki; Matsui, Nobuaki; Akagi, Masaaki

2013-01-01

While gene knockout technology can reveal the roles of proteins in cellular functions, including in mast cells, fetal death due to gene manipulation frequently interrupts experimental analysis. We generated mast cells from mouse fetal liver (FLMC), and compared the fundamental functions of FLMC with those of bone marrow-derived mouse mast cells (BMMC). Under electron microscopy, numerous small and electron-dense granules were observed in FLMC. In FLMC, the expression levels of a subunit of the FcεRI receptor and degranulation by IgE cross-linking were comparable with BMMC. By flow cytometry we observed surface expression of c-Kit prior to that of FcεRI on FLMC, although on BMMC the expression of c-Kit came after FcεRI. The surface expression levels of Sca-1 and c-Kit, a marker of putative mast cell precursors, were slightly different between bone marrow cells and fetal liver cells, suggesting that differentiation stage or cell type are not necessarily equivalent between both lineages. Moreover, this indicates that phenotypically similar mast cells may not have undergone an identical process of differentiation. By comprehensive analysis using the next generation sequencer, the same frequency of gene expression was observed for 98.6% of all transcripts in both cell types. These results indicate that FLMC could represent a new and useful tool for exploring mast cell differentiation, and may help to elucidate the roles of individual proteins in the function of mast cells where gene manipulation can induce embryonic lethality in the mid to late stages of pregnancy. PMID:23573287

Differential transcriptional activation by human T-cell leukemia virus type 1 Tax mutants is mediated by distinct interactions with CREB binding protein and p300.

PubMed

Bex, F; Yin, M J; Burny, A; Gaynor, R B

1998-04-01

The human T-cell leukemia virus type 1 Tax protein transforms human T lymphocytes, which can lead to the development of adult T-cell leukemia. Tax transformation is related to its ability to activate gene expression via the ATF/CREB and the NF-kappaB pathways. Transcriptional activation of these pathways is mediated by the actions of the related coactivators CREB binding protein (CBP) and p300. In this study, immunocytochemistry and confocal microscopy were used to localize CBP and p300 in cells expressing wild-type Tax or Tax mutants that are able to selectively activate gene expression from either the NF-kappaB or ATF/CREB pathway. Wild-type Tax colocalized with both CBP and p300 in nuclear bodies which also contained ATF-1 and the RelA subunit of NF-kappaB. However, a Tax mutant that selectively activates gene expression from only the ATF/CREB pathway colocalized with CBP but not p300, while a Tax mutant that selectively activates gene expression from only the NF-kappaB pathway colocalized with p300 but not CBP. In vitro and in vivo protein interaction studies indicated that the integrity of two independent domains of Tax delineated by these mutants was involved in the direct interaction of Tax with either CBP or p300. These studies are consistent with a model in which activation of either the NF-kappaB or the ATF/CREB pathway by specific Tax mutants is mediated by distinct interactions with related coactivator proteins.

Downregulation of KCNQ5 expression in the rat pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia.

PubMed

Zimmer, Julia; Takahashi, Toshiaki; Hofmann, Alejandro D; Puri, Prem

2017-05-01

Pulmonary hypertension (PH) is a common complication of congenital diaphragmatic hernia (CDH). Voltage-gated potassium channels KCNQ1, KCNQ4, and KCNQ5 are expressed by rodent pulmonary artery smooth muscle cells, contributing to their vascular tone. We hypothesized that KCNQ1, KCNQ4, and KCNQ5 expression is altered in the pulmonary vasculature of nitrofen-induced CDH rats. After ethical approval (REC913b), time-pregnant rats received nitrofen or vehicle on gestational day (D)9. D21 fetuses were divided into CDH and control group (n=22). QRT-PCR and western blotting were performed to determine gene and protein expression of KCNQ1, KCNQ4, and KCNQ5. Confocal microscopy was used to detect these proteins in the pulmonary vasculature. Relative mRNA level of KCNQ5 (p=0.025) was significantly downregulated in CDH lungs compared to controls. KCNQ1 (p=0.052) and KCNQ4 (p=0.574) expression was not altered. Western blotting confirmed the decreased pulmonary KCNQ5 protein expression in CDH lungs. Confocal-microscopy detected a markedly diminished KCNQ5 expression in pulmonary vasculature of CDH fetuses. Downregulated pulmonary expression of KCNQ5 in CDH lungs suggests that this potassium channel may play an important role in the development of PH in this model. KCNQ5 channel activator drugs may be a potential therapeutic target for the treatment of PH in CDH. 2b (Centre for Evidence-Based Medicine, Oxford). Copyright © 2017. Published by Elsevier Inc.

Expanding the genetic toolbox for Leptospira species by generation of fluorescent bacteria.

PubMed

Aviat, Florence; Slamti, Leyla; Cerqueira, Gustavo M; Lourdault, Kristel; Picardeau, Mathieu

2010-12-01

Our knowledge of the genetics and molecular basis of the pathogenesis associated with Leptospira, in comparison to those of other bacterial species, is very limited. An improved understanding of pathogenic mechanisms requires reliable genetic tools for functional genetic analysis. Here, we report the expression of gfp and mRFP1 genes under the control of constitutive spirochetal promoters in both saprophytic and pathogenic Leptospira strains. We were able to reliably measure the fluorescence of Leptospira by fluorescence microscopy and a fluorometric microplate reader-based assay. We showed that the expression of the gfp gene had no significant effects on growth in vivo and pathogenicity in L. interrogans. We constructed an expression vector for L. biflexa that contains the lacI repressor, an inducible lac promoter, and gfp as the reporter, demonstrating that the lac system is functional in Leptospira. Green fluorescent protein (GFP) expression was induced by the addition of isopropyl-β-d-thiogalactopyranoside (IPTG) in L. biflexa transformants harboring the expression vector. Finally, we showed that GFP can be used as a reporter to assess promoter activity in different environmental conditions. These results may facilitate further advances for studying the genetics of Leptospira spp.

Actin expression in trypanosomatids (Euglenozoa: Kinetoplastea)

PubMed Central

Souza, Ligia Cristina Kalb; Pinho, Rosana Elisa Gonçalves Gonçalves; Lima, Carla Vanessa de Paula; Fragoso, Stênio Perdigão; Soares, Maurilio José

2013-01-01

Heteroxenic and monoxenic trypanosomatids were screened for the presence of actin using a mouse polyclonal antibody produced against the entire sequence of the Trypanosoma cruzi actin gene, encoding a 41.9 kDa protein. Western blot analysis showed that this antibody reacted with a polypeptide of approximately 42 kDa in the whole-cell lysates of parasites targeting mammals (T. cruzi, Trypanosoma brucei and Leishmania major), insects (Angomonas deanei, Crithidia fasciculata, Herpetomonas samuelpessoai and Strigomonas culicis) and plants (Phytomonas serpens). A single polypeptide of approximately 42 kDa was detected in the whole-cell lysates of T. cruzi cultured epimastigotes, metacyclic trypomastigotes and amastigotes at similar protein expression levels. Confocal microscopy showed that actin was expressed throughout the cytoplasm of all the tested trypanosomatids. These data demonstrate that actin expression is widespread in trypanosomatids. PMID:23903980

Isolation of the three grape sub-lineages of B-class MADS-box TM6, PISTILLATA and APETALA3 genes which are differentially expressed during flower and fruit development.

PubMed

Poupin, María Josefina; Federici, Fernán; Medina, Consuelo; Matus, José Tomás; Timmermann, Tania; Arce-Johnson, Patricio

2007-12-01

The B class of MADS-box floral homeotic genes specifies petal and stamen identity in angiosperms. While this group is one of the most studied in herbaceous plant species, it has remained largely uncharacterized in woody species such as grapevine. Although the B class PI/GLO and AP3/DEF clades have been extensively characterized in model species, the role of the TM6 subgroup within the AP3 clade is not completely understood, since it is absent in Arabidopsis thaliana. In this study, the coding regions of VvTM6 and VvAP3 and the genomic sequence of VvPI, were cloned. VvPI and AtPI were confirmed to be functional homologues by means of complementation of the pi Arabidopsis mutant. Expression analysis revealed that VvPI and VvAP3 transcripts are restricted almost exclusively to inflorescences, although VvPI was detected at low levels in leaves and roots. VvTM6 expresses throughout the plant, with higher levels in flowers and berries. A detailed chronological study of grape flower progression by light microscopy and temporal expression analysis throughout early and late developmental stages, revealed that VvPI expression increases during pollen maturation and decreases between the events of pollination and fertilization, before the cap fall. On the other hand, VvTM6 is expressed in the last stage of anther development. Specific expression of VvAP3 and VvPI was detected in petals and stamens within the flower, while VvTM6 was also expressed in carpels. Moreover, this work provides the first evidence for expression of a TM6-like gene throughout fruit growth and ripening. Even if these genes belong to the same genetic class they could act in different periods and/or tissues during reproductive organ development.

Probiotic Lactobacillus sp. inhibit growth, biofilm formation and gene expression of caries-inducing Streptococcus mutans.

PubMed

Wasfi, Reham; Abd El-Rahman, Ola A; Zafer, Mai M; Ashour, Hossam M

2018-03-01

Streptococcus mutans contributes significantly to dental caries, which arises from homoeostasic imbalance between host and microbiota. We hypothesized that Lactobacillus sp. inhibits growth, biofilm formation and gene expression of Streptococcus mutans. Antibacterial (agar diffusion method) and antibiofilm (crystal violet assay) characteristics of probiotic Lactobacillus sp. against Streptococcus mutans (ATCC 25175) were evaluated. We investigated whether Lactobacillus casei (ATCC 393), Lactobacillus reuteri (ATCC 23272), Lactobacillus plantarum (ATCC 14917) or Lactobacillus salivarius (ATCC 11741) inhibit expression of Streptococcus mutans genes involved in biofilm formation, quorum sensing or stress survival using quantitative real-time polymerase chain reaction (qPCR). Growth changes (OD600) in the presence of pH-neutralized, catalase-treated or trypsin-treated Lactobacillus sp. supernatants were assessed to identify roles of organic acids, peroxides and bacteriocin. Susceptibility testing indicated antibacterial (pH-dependent) and antibiofilm activities of Lactobacillus sp. against Streptococcus mutans. Scanning electron microscopy revealed reduction in microcolony formation and exopolysaccharide structural changes. Of the oral normal flora, L. salivarius exhibited the highest antibiofilm and peroxide-dependent antimicrobial activities. All biofilm-forming cells treated with Lactobacillus sp. supernatants showed reduced expression of genes involved in exopolysaccharide production, acid tolerance and quorum sensing. Thus, Lactobacillus sp. can inhibit tooth decay by limiting growth and virulence properties of Streptococcus mutans. © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Impacts of ocean acidification on gene expression and biomineralisation in the Pacific oyster Crassostrea gigas Thunberg, 1793

NASA Astrophysics Data System (ADS)

Bagusche, F.; Pouvreau, S.; Trueman, C.; Long, S.; Hauton, C.

2012-04-01

The published evidence of impacts of ocean acidification and on marine calcifiers has emphasized the need to understand the molecular mechanisms of biomineralisation. Crassostrea gigas is an ideal organism to examine these processes as: 1) the hatchery rearing of larval stages is well constrained, 2) studies have established an ontogenetic switch in deposition of carbonate polymorphs from aragonite in larval shells to calcite in adults and 3) it is a globally-important commercial species. Research summarized in this presentation will identify some of the molecular mechanisms involved in calcification processes during ontogeny of Crassostrea gigas, as well as possible impacts of changes in environmental conditions such as temperature and pH. Data will be presented from a quantitative real-time PCR study of the changes in gene expression during development in different environments. Additionally scanning electron microscopy and infrared spectroscopy analyses of shell microstructures and composition will be summarised to correlate changes in gene expression with end-point differences in shell structure. Preliminary results suggest that changes in the environmental conditions lead to differences in expression patterns of genes involved in biomineralisation processes. The combined effects of ambient seawater temperature and low pH show the greatest negative effect on larval shell development, identified as malformations, eroded shell surfaces and a significant decrease in shell size. However, the effect of higher seawater temperature seems to amend the effects of ocean acidification on larval shell development.

Characterization of tail sheath protein of giant bacteriophage phiKZ Pseudomonas aeruginosa

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

Kurochkina, Lidia P., E-mail: lpk@ibch.r; Aksyuk, Anastasia A.; Sachkova, Maria Yu.

2009-12-20

The tail sheath protein of giant bacteriophage phiKZ Pseudomonas aeruginosa encoded by gene 29 was identified and its expression system was developed. Localization of the protein on the virion was confirmed by immunoelectron microscopy. Properties of gene product (gp) 29 were studied by electron microscopy, immunoblotting and limited trypsinolysis. Recombinant gp29 assembles into the regular tubular structures (polysheaths) of variable length. Trypsin digestion of gp29 within polysheaths or extended sheath of virion results in specific cleavage of the peptide bond between Arg135 and Asp136. However, this cleavage does not affect polymeric structure of polysheaths, sheaths and viral infectivity. Digestion bymore » trypsin of the C-truncated gp29 mutant, lacking the ability to self-assemble, results in formation of a stable protease-resistant fragment. Although there is no sequence homology of phiKZ proteins to proteins of other bacteriophages, some characteristic biochemical properties of gp29 revealed similarities to the tail sheath protein of bacteriophage T4.« less

High-throughput microfluidics to control and measure signaling dynamics in single yeast cells

PubMed Central

Hansen, Anders S.; Hao, Nan; O'Shea, Erin K.

2015-01-01

Microfluidics coupled to quantitative time-lapse fluorescence microscopy is transforming our ability to control, measure, and understand signaling dynamics in single living cells. Here we describe a pipeline that incorporates multiplexed microfluidic cell culture, automated programmable fluid handling for cell perturbation, quantitative time-lapse microscopy, and computational analysis of time-lapse movies. We illustrate how this setup can be used to control the nuclear localization of the budding yeast transcription factor Msn2. Using this protocol, we generate oscillations of Msn2 localization and measure the dynamic gene expression response of individual genes in single cells. The protocol allows a single researcher to perform up to 20 different experiments in a single day, whilst collecting data for thousands of single cells. Compared to other protocols, the present protocol is relatively easy to adopt and higher-throughput. The protocol can be widely used to control and monitor single-cell signaling dynamics in other signal transduction systems in microorganisms. PMID:26158443

Nature and mechanisms of hepatocyte apoptosis induced by D-galactosamine/lipopolysaccharide challenge in mice.

PubMed

Wu, Yi-Hang; Hu, Shao-Qing; Liu, Jun; Cao, Hong-Cui; Xu, Wei; Li, Yong-Jun; Li, Lan-Juan

2014-06-01

Apoptosis plays a role in the normal development of liver. However, overactivation thereof may lead to hepatocellular damage. The aim of this study was to assess D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced hepatocyte apoptotic changes in mice and clarify the mechanisms involved in this process. DNA ladder detection was employed to determine the induction condition of hepatic apoptosis. An initial test indicated that typical hepatocyte apoptosis was observed at 6-10 h after the intraperitoneal injection of D-GalN (700 mg/kg) and LPS (10 µg/kg). Subsequently, we evaluated hepatocyte apoptosis at 8 h after administering D-GalN/LPS by histopathological analysis, terminal deoxynucleotidyl transferase-mediated dUTP nick end‑labeling (TUNEL) detection, flow cytometry and electron microscopy analysis. To clarify the apoptosis-related gene expression, the expression levels of tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1), caspase-3, and Fas/Fas ligand (FasL) were determined by serum enzyme immunoassay, immunohistochemistry and western blot analysis. Strong apoptotic positive signals following D-GalN/LPS injection were observed from the results of the serum analysis, histopathological and immunohistochemical analyses, DNA ladder detection, TUNEL detection, flow cytometry and electron microscopy analysis. Additionally, apoptotic hepatocytes were mainly at the late stage of cell apoptosis. The expression of TNF-α, TGF-β1, caspase-3 and Fas/FasL was significantly increased. In conclusion, this study evaluated the D-GalN/LPS-induced hepatocyte apoptotic changes and clarified the apoptosis-related gene expression in mice. The hepatocyte apoptosis induced by D-GalN/LPS may be mainly regulated by the death receptor pathway. TGF-β signaling pathway may also play a vital role in this process of hepatocyte apoptosis.

Tubular overexpression of Gremlin in transgenic mice aggravates renal damage in diabetic nephropathy.

PubMed

Marchant, Vanessa; Droguett, Alejandra; Valderrama, Graciela; Burgos, M Eugenia; Carpio, Daniel; Kerr, Bredford; Ruiz-Ortega, Marta; Egido, Jesús; Mezzano, Sergio

2015-09-15

Diabetic nephropathy (DN) is currently a leading cause of end-stage renal failure worldwide. Gremlin was identified as a gene differentially expressed in mesangial cells exposed to high glucose and in experimental diabetic kidneys. We have described that Gremlin is highly expressed in biopsies from patients with diabetic nephropathy, predominantly in areas of tubulointerstitial fibrosis. In streptozotocin (STZ)-induced experimental diabetes, Gremlin deletion using Grem1 heterozygous knockout mice or by gene silencing, ameliorates renal damage. To study the in vivo role of Gremlin in renal damage, we developed a diabetic model induced by STZ in transgenic (TG) mice expressing human Gremlin in proximal tubular epithelial cells. The albuminuria/creatinuria ratio, determined at week 20 after treatment, was significantly increased in diabetic mice but with no significant differences between transgenic (TG/STZ) and wild-type mice (WT/STZ). To assess the level of renal damage, kidney tissue was analyzed by light microscopy (periodic acid-Schiff and Masson staining), electron microscopy, and quantitative PCR. TG/STZ mice had significantly greater thickening of the glomerular basement membrane, increased mesangial matrix, and podocytopenia vs. WT/STZ. At the tubulointerstitial level, TG/STZ showed increased cell infiltration and mild interstitial fibrosis. In addition, we observed a decreased expression of podocin and overexpression of monocyte chemoattractant protein-1 and fibrotic-related markers, including transforming growth factor-β1, Col1a1, and α-smooth muscle actin. Together, these results show that TG mice overexpressing Gremlin in renal tubules develop greater glomerular and tubulointerstitial injury in response to diabetic-mediated damage and support the involvement of Gremlin in diabetic nephropathy. Copyright © 2015 the American Physiological Society.

Mitochondrial Gene Therapy: Advances in Mitochondrial Gene Cloning, Plasmid Production, and Nanosystems Targeted to Mitochondria.

PubMed

Coutinho, Eduarda; Batista, Cátia; Sousa, Fani; Queiroz, João; Costa, Diana

2017-03-06

Mitochondrial gene therapy seems to be a valuable and promising strategy to treat mitochondrial disorders. The use of a therapeutic vector based on mitochondrial DNA, along with its affinity to the site of mitochondria, can be considered a powerful tool in the reestablishment of normal mitochondrial function. In line with this and for the first time, we successfully cloned the mitochondrial gene ND1 that was stably maintained in multicopy pCAG-GFP plasmid, which is used to transform E. coli. This mitochondrial-gene-based plasmid was encapsulated into nanoparticles. Furthermore, the functionalization of nanoparticles with polymers, such as cellulose or gelatin, enhances their overall properties and performance for gene therapy. The fluorescence arising from rhodamine nanoparticles in mitochondria and a fluorescence microscopy study show pCAG-GFP-ND1-based nanoparticles' cell internalization and mitochondria targeting. The quantification of GFP expression strongly supports this finding. This work highlights the viability of gene therapy based on mitochondrial DNA instigating further in vitro research and clinical translation.

The equine herpesvirus-1 IR3 gene that lies antisense to the sole immediate-early (IE) gene is trans-activated by the IE protein, and is poorly expressed to a protein

PubMed Central

Ahn, Byung Chul; Breitenbach, Jonathan E.; Kim, Seong K.; O’Callaghan, Dennis J.

2007-01-01

The unique IR3 gene of equine herpesvirus 1 (EHV-1) is expressed as a late 1.0-kb transcript. Previous studies confirmed the IR3 transcription initiation site and tentatively identified other cis-acting elements specific to IR3 such as a TATA box, a 443 base pair 5′untranslated region (UTR), a 285 base pair open reading frame (ORF) and a poly adenylation (A) signal (Holden et al., 1992 DNA Seq 3, 143-52). Transient transfection assays revealed that the IR3 promoter is strongly trans-activated by the IE protein (IEP) and that coexpression of the IEP with the early EICP0 and IR4 regulatory proteins results in maximal trans-activation of the IR3 promoter. Gel shift assays revealed that the IEP directly binds to the IR3 promoter region. Western blot analysis showed that the IR3 protein produced in E. coli was detected by antibodies to IR3 synthetic peptides; however, the IR3 protein was not detected in EHV-1 infected cell extracts by these same anti-IR3 antibodies, even though the IR3 transcript was detected by northern blot. These findings suggest that the IR3 may not be expressed to a protein. Expression of an IR3/GFP fusion gene was not observed, but expression of a GFP/IR3 fusion gene was detected by fluorescent microscopy. In further attempts to detect the IR3/GFP fusion protein using anti-GFP antibody, western blot analysis showed that the IR3/GFP fusion protein was not detected in vivo. Interestingly, a truncated form of the GFP/IR3 protein was synthesized from the GFP/IR3 fusion gene. However, GFP/IR3 and IR3/GFP fusion proteins of the predicted sizes were synthesized by in vitro coupled transcription and translation of the fusion genes, suggesting poor expression of the IR3 protein in vivo. The possible role of the IR3 transcript in EHV-1 infection is discussed. PMID:17306852

Genes related to antioxidant metabolism are involved in Methylobacterium mesophilicum-soybean interaction.

PubMed

Araújo, Welington Luiz; Santos, Daiene Souza; Dini-Andreote, Francisco; Salgueiro-Londoño, Jennifer Katherine; Camargo-Neves, Aline Aparecida; Andreote, Fernando Dini; Dourado, Manuella Nóbrega

2015-10-01

The genus Methylobacterium is composed of pink-pigmented methylotrophic bacterial species that are widespread in natural environments, such as soils, stream water and plants. When in association with plants, this genus colonizes the host plant epiphytically and/or endophytically. This association is known to promote plant growth, induce plant systemic resistance and inhibit plant infection by phytopathogens. In the present study, we focused on evaluating the colonization of soybean seedling-roots by Methylobacterium mesophilicum strain SR1.6/6. We focused on the identification of the key genes involved in the initial step of soybean colonization by methylotrophic bacteria, which includes the plant exudate recognition and adaptation by planktonic bacteria. Visualization by scanning electron microscopy revealed that M. mesophilicum SR1.6/6 colonizes soybean roots surface effectively at 48 h after inoculation, suggesting a mechanism for root recognition and adaptation before this period. The colonization proceeds by the development of a mature biofilm on roots at 96 h after inoculation. Transcriptomic analysis of the planktonic bacteria (with plant) revealed the expression of several genes involved in membrane transport, thus confirming an initial metabolic activation of bacterial responses when in the presence of plant root exudates. Moreover, antioxidant genes were mostly expressed during the interaction with the plant exudates. Further evaluation of stress- and methylotrophic-related genes expression by qPCR showed that glutathione peroxidase and glutathione synthetase genes were up-regulated during the Methylobacterium-soybean interaction. These findings support that glutathione (GSH) is potentially a key molecule involved in cellular detoxification during plant root colonization. In addition to methylotrophic metabolism, antioxidant genes, mainly glutathione-related genes, play a key role during soybean exudate recognition and adaptation, the first step in bacterial colonization.

Biomimetic High Density Lipoprotein Nanoparticles For Nucleic Acid Delivery

PubMed Central

McMahon, Kaylin M.; Mutharasan, R. Kannan; Tripathy, Sushant; Veliceasa, Dorina; Bobeica, Mariana; Shumaker, Dale K.; Luthi, Andrea J.; Helfand, Brian T.; Ardehali, Hossein; Mirkin, Chad A.; Volpert, Olga; Thaxton, C. Shad

2014-01-01

We report a gold nanoparticle-templated high density lipoprotein (HDL AuNP) platform for gene therapy which combines lipid-based nucleic acid transfection strategies with HDL biomimicry. For proof-of-concept, HDL AuNPs are shown to adsorb antisense cholesterylated DNA. The conjugates are internalized by human cells, can be tracked within cells using transmission electron microscopy (TEM), and regulate target gene expression. Overall, the ability to directly image the AuNP core within cells, the chemical tailorability of the HDL AuNP platform, and the potential for cell-specific targeting afforded by HDL biomimicry make this platform appealing for nucleic acid delivery. PMID:21319839

Systematic characterization of maturation time of fluorescent proteins in living cells

PubMed Central

Balleza, Enrique; Kim, J. Mark; Cluzel, Philippe

2017-01-01

Slow maturation time of fluorescent proteins limits accurate measurement of rapid gene expression dynamics and effectively reduces fluorescence signal in growing cells. We used high-precision time-lapse microscopy to characterize, at two different temperatures in E. coli, the maturation kinetics of 50 FPs that span the visible spectrum. We identified fast-maturing FPs that yield the highest signal-to-noise ratio and temporal resolution in individual growing cells. PMID:29320486

Electron microscopy of lamin and the nuclear lamina in Caenorhabditis elegans.

PubMed

Cohen, Merav; Santarella, Rachel; Wiesel, Naama; Mattaj, Iain; Gruenbaum, Yosef

2008-01-01

The nuclear lamina is found between the inner nuclear membrane and the peripheral chromatin. Lamins are the main components of the nuclear lamina, where they form protein complexes with integral proteins of the inner nuclear membrane, transcriptional regulators, histones and chromatin modifiers. Lamins are required for mechanical stability, chromatin organization, Pol II transcription, DNA replication, nuclear assembly, and nuclear positioning. Mutations in human lamins cause at least 13 distinct human diseases, collectively termed laminopathies, affecting muscle, adipose, bone, nerve and skin cells, and range from muscular dystrophies to accelerated aging. Caenorhabditis elegans has unique advantages in studying lamins and nuclear lamina genes including low complexity of lamina genes and the unique ability of bacterially expressed C. elegans lamin protein to form stable 10 nm fibers. In addition, transgenic techniques, simple application of RNA interference, sophisticated genetic analyses, and the production of a large collection of mutant lines, all make C. elegans especially attractive for studying the functions of its nuclear lamina genes. In this chapter we will include a short review of our current knowledge of nuclear lamina in C. elegans and will describe electron microscopy techniques used for their analyses.

Porous titanium and Ti-35Nb alloy: effects on gene expression of osteoblastic cells derived from human alveolar bone.

PubMed

do Prado, Renata Falchete; Rabêlo, Sylvia Bicalho; de Andrade, Dennia Perez; Nascimento, Rodrigo Dias; Henriques, Vinicius André Rodrigues; Carvalho, Yasmin Rodarte; Cairo, Carlos Alberto Alves; de Vasconcellos, Luana Marotta Reis

2015-11-01

Tests on titanium alloys that possess low elastic modulus, corrosion resistance and minimal potential toxicity are ongoing. This study aimed to evaluate the behavior of human osteoblastic cells cultured on dense and porous Titanium (Ti) samples comparing to dense and porous Ti-35 Niobium (Ti-35Nb) samples, using gene expression analysis. Scanning electronic microscopy confirmed surface porosity and pore interconnectivity and X-ray diffraction showed titanium beta-phase stabilization in Ti-35Nb alloy. There were no differences in expression of transforming growth factor-β, integrin-β1, alkaline phosphatase, osteopontin, macrophage colony stimulating factor, prostaglandin E synthase, and apolipoprotein E regarding the type of alloy, porosity and experimental period. The experimental period was a significant factor for the markers: bone sialoprotein II and interleukin 6, with expression increasing over time. Porosity diminished Runt-related transcription factor-2 (Runx-2) expression. Cells adhering to the Ti-35Nb alloy showed statistically similar expression to those adhering to commercially pure Ti grade II, for all the markers tested. In conclusion, the molecular mechanisms of interaction between human osteoblasts and the Ti-35Nb alloy follow the principal routes of osseointegration of commercially pure Ti grade II. Porosity impaired the route of transcription factor Runx-2.

Expression of the Drosophila homeobox gene, Distal-less supports an ancestral role in neural development

PubMed Central

Plavicki, Jessica S.; Squirrell, Jayne M.; Eliceiri, Kevin W.; Boekhoff-Falk, Grace

2015-01-01

Background Distal-less (Dll) encodes a homeodomain transcription factor expressed in developing appendages of organisms throughout metazoan phylogeny. Based on earlier observations in the limbless nematode Caenorhabditis elegans and the primitive chordate amphioxus, it was proposed that Dll had an ancestral function in nervous system development. Consistent with this hypothesis, Dll is necessary for the development of both peripheral and central components of the Drosophila olfactory system. Furthermore, vertebrate homologs of Dll, the Dlx genes, play critical roles in mammalian brain development. Results Using fluorescent immunohistochemistry of fixed samples and multiphoton microscopy of living Drosophila embryos we show that Dll is expressed in the embryonic, larval and adult CNS and PNS in embryonic and larval neurons, brain and ventral nerve cord (VNC) glia, as well as in PNS structures associated with chemosensation. In adult flies, Dll expression is expressed in the optic lobes, central brain regions and the antennal lobes. Conclusions Characterization of Dll expression in the developing nervous system supports a role of Dll in neural development and function and establishes an important basis for determining the specific functional roles of Dll in Drosophila development and for comparative studies of Drosophila Dll functions with those of its vertebrate counterparts. PMID:26472170

Differential association of S100A9, an inflammatory marker, and p53, a cell cycle marker, expression with epicardial adipocyte size in patients with cardiovascular disease.

PubMed

Agra, Rosa María; Fernández-Trasancos, Ángel; Sierra, Juan; González-Juanatey, José Ramón; Eiras, Sonia

2014-10-01

S100A9 (calgranulin B) has inflammatory and oxidative stress properties and was found to be associated with atherosclerosis and obesity. One of the proteins that can regulate S100A9 transcription is p53, which is involved in cell cycle, apoptosis and adipogenesis. Thus, it triggers adipocyte enlargement and finally obesity. Because epicardial adipose tissue (EAT) volume and thickness is related to coronary artery disease (CAD), we studied the gene expression of this pathway in patients with cardiovascular disease and its association with obesity. Adipocytes and stromal cells from EAT and subcutaneous adipose tissue (SAT) from 48 patients who underwent coronary artery bypass graft and/or valve replacement were obtained after collagenase digestion and differential centrifugation. The expression levels of the involved genes on adipogenesis and cell cycle like fatty acid-binding protein (FABP) 4, retinol-binding protein (RBP)4, p53 and S100A9 were determined by real-time polymerase chain reaction (PCR). Adipocyte diameter was measured by optical microscopy. We found that epicardial adipocytes expressed significantly lower levels of adipogenic genes (FABP4 and RBP4) and cell cycle-related genes (S100A9 and p53) than subcutaneous adipocytes. However, in obese patients, upregulation of adipogenic and cell cycle-related genes in subcutaneous and epicardial adipocytes, respectively, was observed. The enlargement of adipocyte size was related to FABP4, S100A9 and p53 expression levels in stromal cells. But only the p53 association was maintained in epicardial stromal cells from obese patients (p=0.003). The expression of p53, but not S100A9, in epicardial stromal cells is related to adipocyte enlargement in obese patients with cardiovascular disease. These findings suggest new mechanisms for understanding the relationship between epicardial fat thickness, obesity and cardiovascular disease.

CER4 Encodes an Alcohol-Forming Fatty Acyl-Coenzyme A Reductase Involved in Cuticular Wax Production in Arabidopsis1[W

PubMed Central

Rowland, Owen; Zheng, Huanquan; Hepworth, Shelley R.; Lam, Patricia; Jetter, Reinhard; Kunst, Ljerka

2006-01-01

A waxy cuticle that serves as a protective barrier against uncontrolled water loss and environmental damage coats the aerial surfaces of land plants. It is composed of a cutin polymer matrix and waxes. Cuticular waxes are complex mixtures of very-long-chain fatty acids and their derivatives. We report here the molecular cloning and characterization of CER4, a wax biosynthetic gene from Arabidopsis (Arabidopsis thaliana). Arabidopsis cer4 mutants exhibit major decreases in stem primary alcohols and wax esters, and slightly elevated levels of aldehydes, alkanes, secondary alcohols, and ketones. This phenotype suggested that CER4 encoded an alcohol-forming fatty acyl-coenzyme A reductase (FAR). We identified eight FAR-like genes in Arabidopsis that are highly related to an alcohol-forming FAR expressed in seeds of jojoba (Simmondsia chinensis). Molecular characterization of CER4 alleles and genomic complementation revealed that one of these eight genes, At4g33790, encoded the FAR required for cuticular wax production. Expression of CER4 cDNA in yeast (Saccharomyces cerevisiae) resulted in the accumulation of C24:0 and C26:0 primary alcohols. Fully functional green fluorescent protein-tagged CER4 protein was localized to the endoplasmic reticulum in yeast cells by confocal microscopy. Analysis of gene expression by reverse transcription-PCR indicated that CER4 was expressed in leaves, stems, flowers, siliques, and roots. Expression of a β-glucuronidase reporter gene driven by the CER4 promoter in transgenic plants was detected in epidermal cells of leaves and stems, consistent with a dedicated role for CER4 in cuticular wax biosynthesis. CER4 was also expressed in all cell types in the elongation zone of young roots. These data indicate that CER4 is an alcohol-forming FAR that has specificity for very-long-chain fatty acids and is responsible for the synthesis of primary alcohols in the epidermal cells of aerial tissues and in roots. PMID:16980563

Genetic transformation of novel isolates of chicken Lactobacillus bearing probiotic features for expression of heterologous proteins: a tool to develop live oral vaccines

PubMed Central

Mota, Rodrigo M; Moreira, João Luiz S; Souza, Marcelo R; Fátima Horta, M; Teixeira, Santuza MR; Neumann, Elisabeth; Nicoli, Jacques R; Nunes, Álvaro C

2006-01-01

Background The use of lactic acid bacteria as vehicles to delivery antigens to immunize animals is a promising issue. When genetically modified, these bacteria can induce a specific local and systemic immune response against selected pathogens. Gastric acid and bile salts tolerance, production of antagonistic substances against pathogenic microorganisms, and adhesive ability to gut epithelium are other important characteristics that make these bacteria useful for oral immunization. Results Bacteria isolated on de Man, Rogosa and Sharpe medium (MRS) from different gastrointestinal portions of broiler chicks were evaluated for their resistance to artificial gastric acid and bile salts, production of hydrogen peroxide, and cell surface hydrophobicity. Thirty-eight isolates were first typed at species level by PCR amplification of 16S-23S rRNA intergenic spacers using universal primers that anneal within 16S and 23S genes, followed by restriction digestion analyses of PCR amplicons (PCR-ARDRA). An expression cassette was assembled onto the pCR2.1-Topo vector by cloning the promoter, leader peptide, cell wall anchor and terminator sequences derived from the laminin binding S-layer protein gene of L. crispatus strain F5.7 (lbs gene). A sequence encoding the green fluorescent protein (GFP) was inserted as reporter gene, and an erythromycin resistance gene was added as selective marker. All constructs were able to express GFP in the cloning host E. coli XL1-Blue and different Lactobacillus strains as verified by FACS and laser scanning confocal microscopy. Conclusion Lactobacillus isolated from gastrointestinal tract of broiler chickens and selected for probiotic characteristics can be genetically modified by introducing an expression cassette into the lbs locus. The transformed bacteria expressed on its cell wall surface different fluorescent proteins used as reporters of promoter function. It is possible then that similar bacterial model expressing pathogen antigens can be used as live oral vaccines to immunize broilers against infectious diseases. PMID:16396687

Expression of RNA interference triggers from an oncolytic herpes simplex virus results in specific silencing in tumour cells in vitro and tumours in vivo

PubMed Central

2010-01-01

Background Delivery of small interfering RNA (siRNA) to tumours remains a major obstacle for the development of RNA interference (RNAi)-based therapeutics. Following the promising pre-clinical and clinical results with the oncolytic herpes simplex virus (HSV) OncoVEXGM-CSF, we aimed to express RNAi triggers from oncolytic HSV, which although has the potential to improve treatment by silencing tumour-related genes, was not considered possible due to the highly oncolytic properties of HSV. Methods To evaluate RNAi-mediated silencing from an oncolytic HSV backbone, we developed novel replicating HSV vectors expressing short-hairpin RNA (shRNA) or artificial microRNA (miRNA) against the reporter genes green fluorescent protein (eGFP) and β-galactosidase (lacZ). These vectors were tested in non-tumour cell lines in vitro and tumour cells that are moderately susceptible to HSV infection both in vitro and in mice xenografts in vivo. Silencing was assessed at the protein level by fluorescent microscopy, x-gal staining, enzyme activity assay, and western blotting. Results Our results demonstrate that it is possible to express shRNA and artificial miRNA from an oncolytic HSV backbone, which had not been previously investigated. Furthermore, oncolytic HSV-mediated delivery of RNAi triggers resulted in effective and specific silencing of targeted genes in tumour cells in vitro and tumours in vivo, with the viruses expressing artificial miRNA being comprehensibly more effective. Conclusions This preliminary data provide the first demonstration of oncolytic HSV-mediated expression of shRNA or artificial miRNA and silencing of targeted genes in tumour cells in vitro and in vivo. The vectors developed in this study are being adapted to silence tumour-related genes in an ongoing study that aims to improve the effectiveness of oncolytic HSV treatment in tumours that are moderately susceptible to HSV infection and thus, potentially improve response rates seen in human clinical trials. PMID:20836854

In vitro activity of chlorogenic acid against Aspergillus fumigatus biofilm and gliotoxin production.

PubMed

Kong, Jin-Liang; Luo, Jing; Li, Bing; Dong, Bi-Ying; Huang, Hong; Wang, Ke; Wu, Li-Hong; Chen, Yi-Qiang

2017-06-01

Aspergillus ( A .) fumigatus , one of the most common causes of life-threatening fungal infections in immunocompromised patients, shows resistance to antifungal agents as has a high propensity to forming a biofilm. The present study aimed to investigate the effects of chlorogenic acid (CRA) on A. fumigatus biofilm formation and integrity. Confocal laser scanning microscopy was performed to determine the inhibitory effects of CRA against A. fumigatus biofilm formation. Transmission electron microscopy was performed to investigate the ultrastructural changes of A. fumigatus biofilm after CRA exposure. High-performance liquid chromatography and reverse-transcription quantitative PCR were performed to determine the expression of gliotoxin production in biofilm culture. The results showed that CRA at sub-minimum inhibitory concentrations inhibited A. fumigatus biofilm formation. In addition, CRA could decreased the gliotoxin production in the biofilm culture supernatant through inhibiting the expression of master genes involved in gliotoxin biosynthesis. The present study provided useful information for the development of novel strategies to reduce the incidence of A. fumigatus biofilm-associated diseases.

Light-induced c-Fos expression in the mouse suprachiasmatic nucleus: immunoelectron microscopy reveals co-localization in multiple cell types.

PubMed

Castel, M; Belenky, M; Cohen, S; Wagner, S; Schwartz, W J

1997-09-01

Although light is known to regulate the level of c-fos gene expression in the suprachiasmatic nucleus (SCN), the site of an endogenous circadian clock, little is known about the identities of the photically activated cells. We used light-microscopic immunocytochemistry and immunoelectron microscopy to detect c-Fos protein in the SCN of Sabra mice exposed to brief nocturnal light pulses at zeitgeber time 15-16. Stimulation with light pulses that saturated the phase-shifting response of the circadian locomotor rhythm revealed an upper limit to the number of photo-inducible c-Fos cells at about one-fifth of the estimated total SCN cell population. This functionally defined set was morphologically and phenotypically heterogeneous. About 24% could be labelled for vasoactive intestinal polypeptide, 13% for vasopressin-neurophysin, and 7% for glial fibrillary acidic protein. The remaining 56% of c-Fos-positive cells were largely of unknown phenotype, although many were presumptive interneurons, some of which were immunoreactive for nitric oxide synthase.

High Throughput, High Content Screening for Novel Pigmentation Regulators Using a Keratinocyte/Melanocyte Co-culture System

PubMed Central

Lee, Ju Hee; Chen, Hongxiang; Kolev, Vihren; Aull, Katherine H.; Jung, Inhee; Wang, Jun; Miyamoto, Shoko; Hosoi, Junichi; Mandinova, Anna; Fisher, David E.

2014-01-01

Skin pigmentation is a complex process including melanogenesis within melanocytes and melanin transfer to the keratinocytes. To develop a comprehensive screening method for novel pigmentation regulators, we used immortalized melanocytes and keratinocytes in co-culture to screen large numbers of compounds. High-throughput screening plates were subjected to digital automated microscopy to quantify the pigmentation via brightfield microscopy. Compounds with pigment suppression were secondarily tested for their effects on expression of MITF and several pigment regulatory genes, and further validated in terms of non-toxicity to keratinocytes/melanocytes and dose dependent activity. The results demonstrate a high-throughput, high-content screening approach, which is applicable to the analysis of large chemical libraries using a co-culture system. We identified candidate pigmentation inhibitors from 4,000 screened compounds including zoxazolamine, 3-methoxycatechol, and alpha-mangostin, which were also shown to modulate expression of MITF and several key pigmentation factors, and are worthy of further evaluation for potential translation to clinical use. PMID:24438532

Enhancement of Thiamine Biosynthesis in Oil Palm Seedlings by Colonization of Endophytic Fungus Hendersonia toruloidea

PubMed Central

Kamarudin, Amirah N.; Lai, Kok S.; Lamasudin, Dhilia U.; Idris, Abu S.; Balia Yusof, Zetty N.

2017-01-01

Thiamine, or vitamin B1 plays an indispensable role as a cofactor in crucial metabolic reactions including glycolysis, pentose phosphate pathway and the tricarboxylic acid cycle in all living organisms. Thiamine has been shown to play a role in plant adaptation toward biotic and abiotic stresses. The modulation of thiamine biosynthetic genes in oil palm seedlings was evaluated in response to root colonization by endophytic Hendersonia toruloidea. Seven-month-old oil palm seedlings were inoculated with H. toruloidea and microscopic analyses were performed to visualize the localization of endophytic H. toruloidea in oil palm roots. Transmission electron microscopy confirmed that H. toruloidea colonized cortical cells. The expression of thiamine biosynthetic genes and accumulation of total thiamine in oil palm seedlings were also evaluated. Quantitative real-time PCR was performed to measure transcript abundances of four key thiamine biosynthesis genes (THI4, THIC, TH1, and TPK) on days 1, 7, 15, and 30 in response to H. toruloidea colonization. The results showed an increase of up to 12-fold in the expression of all gene transcripts on day 1 post-inoculation. On days 7, 15, and 30 post-inoculation, the relative expression levels of these genes were shown to be downregulated. Thiamine accumulation was observed on day 7 post-colonization and subsequently decreased until day 30. This work provides the first evidence for the enhancement of thiamine biosynthesis by endophytic colonization in oil palm seedlings. PMID:29089959

Enhancement of Thiamine Biosynthesis in Oil Palm Seedlings by Colonization of Endophytic Fungus Hendersonia toruloidea.

PubMed

Kamarudin, Amirah N; Lai, Kok S; Lamasudin, Dhilia U; Idris, Abu S; Balia Yusof, Zetty N

2017-01-01

Thiamine, or vitamin B1 plays an indispensable role as a cofactor in crucial metabolic reactions including glycolysis, pentose phosphate pathway and the tricarboxylic acid cycle in all living organisms. Thiamine has been shown to play a role in plant adaptation toward biotic and abiotic stresses. The modulation of thiamine biosynthetic genes in oil palm seedlings was evaluated in response to root colonization by endophytic Hendersonia toruloidea . Seven-month-old oil palm seedlings were inoculated with H. toruloidea and microscopic analyses were performed to visualize the localization of endophytic H. toruloidea in oil palm roots. Transmission electron microscopy confirmed that H. toruloidea colonized cortical cells. The expression of thiamine biosynthetic genes and accumulation of total thiamine in oil palm seedlings were also evaluated. Quantitative real-time PCR was performed to measure transcript abundances of four key thiamine biosynthesis genes ( THI4 , THIC , TH1 , and TPK ) on days 1, 7, 15, and 30 in response to H. toruloidea colonization. The results showed an increase of up to 12-fold in the expression of all gene transcripts on day 1 post-inoculation. On days 7, 15, and 30 post-inoculation, the relative expression levels of these genes were shown to be downregulated. Thiamine accumulation was observed on day 7 post-colonization and subsequently decreased until day 30. This work provides the first evidence for the enhancement of thiamine biosynthesis by endophytic colonization in oil palm seedlings.

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

Park, Joo-Man; Kim, Tae-Hyun; Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul 120-752

Research highlights: {yields} Insulin-suppression of PEPCK and G6Pase gene expression is counteracted by resveratrol. {yields} Resveratrol upregulates hepatic gluconeogenic genes by attenuating insulin signaling and deacetylating FOXO1, which are SIRT1-independent in the cytosol and SIRT1-dependent in the nucleus, respectively. {yields} Resveratrol increases the binding activity of Foxo1 to the IRE of PEPCK and G6Pase. -- Abstract: During a state of fasting, the blood glucose level is maintained by hepatic gluconeogenesis. SIRT1 is an important metabolic regulator during nutrient deprivation and the liver-specific knockdown of SIRT1 resulted in decreased glucose production. We hypothesize that SIRT1 is responsible for the upregulation ofmore » insulin-suppressed gluconeogenic genes through the deacetylation of FOXO1. Treatment of primary cultured hepatocytes with resveratrol increased insulin-repressed PEPCK and G6Pase mRNA levels, which depend on SIRT1 activity. We found that the resveratrol treatment resulted in a decrease in the phosphorylation of Akt and FOXO1, which are independent of SIRT1 action. Fluorescence microscopy revealed that resveratrol caused the nuclear localization of FOXO1. In the nucleus, FOXO1 is deacetylated by SIRT1, which might make it more accessible to the IRE of the PEPCK and G6Pase promoter, causing an increase in their gene expression. Our results indicate that resveratrol upregulates the expression of gluconeogenic genes by attenuating insulin signaling and by deacetylating FOXO1, which are SIRT1-independent in the cytosol and SIRT1-dependent in the nucleus, respectively.« less

Effects of pCO2 stress on gene expression and biomineralization of developing larvae of the Pacific oyster Crassostrea gigas.

NASA Astrophysics Data System (ADS)

De Wit, P.; Durland, E.; Ventura, A.; Waldbusser, G. G.; Langdon, C. J.

2016-02-01

The high larval mortalities in oyster hatcheries on the US west coast have gotten large media coverage in the past few years, and the link has been made between occurrences of coastal upwelling of deep water with low carbonate ion availability and abnormal shell formation in hatchery larvae. However, the mechanism by which this happens is still not well understood. In the Pacific oyster, numerous genes are known to be involved in biomineralization but little is known about the timing of gene expression in relation to formation of the initial larval shell. In order to study this process, we scanned all expressed larval genes using an RNA-Seq approach over the time interval of initial shell formation in both control and pCO2-stressed conditions. Scanning the expression data for patterns matching observed shell formation rates (see Fig 1), we identified a number of genes potentially involved in shell nucleation, most of which are involved in transmembrane transport or protein binding. In addition, we also identified a set of co-expressed genes likely to be involved in the cellular early shell formation machinery. This study is the first to investigate the genes involved in the initial larval shell formation in the Pacific oyster. We discover a set of 149 genes that are likely involved in this process from a combination of CPL microscopy and RNA-Seq, most of which are involved in ion transport or protein binding. These are the two main processes involved in shell formation. Additionally, we observe an increase in the relative content of wax esters in control larvae after 18 hours, something not seen in the treatment larvae. The reason for this is not quite clear at this point, but it could be speculated that stressed larvae develop slower, thus consuming lipids at a slower rate. Thus, follow-up experiments that study the long-term effects of changed carbonate chemistry on the genetics of Pacific oysters will be critical for future aquaculture efforts.

Genome-Wide Screens Reveal New Gene Products That Influence Genetic Competence in Streptococcus mutans

PubMed Central

O'Brien, Greg; Maricic, Natalie; Kesterson, Alexandria; Grace, Megan

2017-01-01

ABSTRACT A network of genes and at least two peptide signaling molecules tightly control when Streptococcus mutans becomes competent to take up DNA from its environment. Widespread changes in the expression of genes occur when S. mutans is presented with competence signal peptides in vitro, including the increased production of the alternative sigma factor, ComX, which activates late competence genes. Still, the way that gene products that are regulated by competence peptides influence DNA uptake and cellular physiology are not well understood. Here, we developed and employed comprehensive transposon mutagenesis of the S. mutans genome, with a screen to identify mutants that aberrantly expressed comX, coupled with transposon sequencing (Tn-seq) to gain a more thorough understanding of the factors modulating comX expression and progression to the competent state. The screens effectively identified genes known to affect competence, e.g., comR, comS, comD, comE, cipB, clpX, rcrR, and ciaH, but disclosed an additional 20 genes that were not previously competence associated. The competence phenotypes of mutants were characterized, including by fluorescence microscopy to determine at which stage the mutants were impaired for comX activation. Among the novel genes studied were those implicated in cell division, the sensing of cell envelope stress, cell envelope biogenesis, and RNA stability. Our results provide a platform for determining the specific chemical and physical cues that are required for genetic competence in S. mutans, while highlighting the effectiveness of using Tn-seq in S. mutans to discover and study novel biological processes. IMPORTANCE Streptococcus mutans acquires DNA from its environment by becoming genetically competent, a physiologic state triggered by cell-cell communication using secreted peptides. Competence is important for acquiring novel genetic traits and has a strong influence on the expression of virulence-associated traits of S. mutans. Here, we used transposon mutagenesis and genomic technologies to identify novel genes involved in competence development. In addition to identifying genes previously known to be required for comX expression, 20 additional genes were identified and characterized. The findings create opportunities to diminish the pathogenic potential of S. mutans, while validating technologies that can rapidly advance our understanding of the physiology, biology, and genetics of S. mutans and related pathogens. PMID:29109185

Genome-wide screens reveal new gene products that influence genetic competence in Streptococcus mutans.

PubMed

Shields, Robert C; O'Brien, Greg; Maricic, Natalie; Kesterson, Alexandria; Grace, Megan; Hagen, Stephen J; Burne, Robert A

2017-11-06

A network of genes and at least two peptide signaling molecules tightly control when Streptococcus mutans becomes competent to take up DNA from its environment. Widespread changes in the expression of genes occur when S. mutans is presented with competence signal peptides in vitro , including increased production of the alternative sigma factor, ComX, which activates late competence genes. Still, the way that gene products that are regulated by competence peptides influence DNA uptake and cellular physiology are not well understood. Here, we developed and employed comprehensive transposon mutagenesis of the S. mutans genome with a screen to identify mutants that aberrantly expressed comX , coupled with transposon sequencing (Tn-seq) to gain a more thorough understanding of the factors modulating comX expression and progression to the competent state. The screens effectively identified genes known to affect competence, e.g. comR , comS , comD , comE , cipB , clpX , rcrR , ciaH , but disclosed an additional 20 genes that were not previously competence-associated. The competence phenotypes of mutants were characterized, including using fluorescence microscopy to determine at which stage the mutants were impaired for comX activation. Among the novel genes studied were those implicated in cell division, sensing of cell envelope stress, cell envelope biogenesis, and RNA stability. Our results provide a platform for determining the specific chemical and physical cues that are required for genetic competence in S. mutans , while highlighting the effectiveness of using Tn-seq in S. mutans to discover and study novel biological processes. IMPORTANCE Streptococcus mutans acquires DNA from its environment by becoming genetically competent, a physiologic state triggered by cell-cell communication using secreted peptides. Competence is important for acquiring novel genetic traits and has a strong influence on the expression of virulence-associated traits of S. mutans Here, we used transposon mutagenesis and genomic technologies to identify novel genes involved in competence development. In addition to identifying genes previously known to be required for comX expression, 20 additional genes were identified and characterized. The findings create opportunities to diminish the pathogenic potential of S. mutans , while validating technologies that can rapidly advance our understanding of the physiology, biology and genetics of S. mutans and related pathogens. Copyright © 2017 American Society for Microbiology.

Microscopy and bioinformatic analyses of lipid metabolism implicate a sporophytic signaling network supporting pollen development in Arabidopsis.

PubMed

Wang, Yixing; Wu, Hong; Yang, Ming

2008-07-01

The Arabidopsis sporophytic tapetum undergoes a programmed degeneration process to secrete lipid and other materials to support pollen development. However, the molecular mechanism regulating the degeneration process is unknown. To gain insight into this molecular mechanism, we first determined that the most critical period for tapetal secretion to support pollen development is from the vacuolate microspore stage to the early binucleate pollen stage. We then analyzed the expression of enzymes responsible for lipid biosynthesis and degradation with available in-silico data. The genes for these enzymes that are expressed in the stamen but not in the concurrent uninucleate microspore and binucleate pollen are of particular interest, as they presumably hold the clues to unique molecular processes in the sporophytic tissues compared to the gametophytic tissue. No gene for lipid biosynthesis but a single gene encoding a patatin-like protein likely for lipid mobilization was identified based on the selection criterion. A search for genes co-expressed with this gene identified additional genes encoding typical signal transduction components such as a leucine-rich repeat receptor kinase, an extra-large G-protein, other protein kinases, and transcription factors. In addition, proteases, cell wall degradation enzymes, and other proteins were also identified. These proteins thus may be components of a signaling network leading to degradation of a broad range of cellular components. Since a broad range of degradation activities is expected to occur only in the tapetal degeneration process at this stage in the stamen, it is further hypothesized that the signaling network acts in the tapetal degeneration process.

Anti-tumor function of double-promoter regulated adenovirus carrying SEA gene, in the treatment of bladder cancer.

PubMed

Hu, Jianpeng; Xuan, Xujun; Han, Conghui; Hao, Lin; Zhang, Peiying; Chen, Meng; He, Houguang; Fan, Tao; Dong, Binzheng

2012-03-01

To construct an adenovirus carrying SEA gene and regulated by telomerase reverse transcriptase (hTERT) and hypoxia-inducible factor (HIF) promoters and investigate its anti-tumor function in vitro, as well as its role in lymphocyte production. hTERT and HIF genes were cloned into adenovirus E1A and E1B shuttle plasmids. The control vector for SEA gene expression is under the regulation of CMV and SV40 promoters. Double regulation was obtained through homologous recombination. The positive clones of replicable adenovirus H2-SEA-Ad were selected by plaque assay. The adenovirus was purified, titrated, and DNA was verified by PCR. The obtained virus was used to infect EJ bladder tumor cells and the SEA Mrna, and protein expression was measured by RT-PCR, western blot, and immunofluorescence microscopy, respectively. Co-culture of lymphocytes and tumor cells was observed dynamically under microscope. The construction of shuttle plasmid p315-CSS-SEA was confirmed by PCR and DNA sequencing. Insertion of superantigen SEA gene in adenovirus (H2-SEA-Ad.SEA) was obtained by homologous recombination. In lymphocytes and tumor cell co-culture, the number of viable tumor cells in test groups was significantly lower than that in control group after 12, 24, and 48 h of treatment. Production of interleukin-2, interleukin-4, and tumor necrosis factor were higher in test groups than in control group. Expression of SEA gene in bladder tumor cells by adenoviral vector caused reduced tumor cell proliferation, as well as stimulation of inflammatory cytokine productions in co-cultures with lymphocytes.

Transposon mutagenesis of type III group B Streptococcus: correlation of capsule expression with virulence.

PubMed

Rubens, C E; Wessels, M R; Heggen, L M; Kasper, D L

1987-10-01

The capsular polysaccharide of type III group B Streptococcus (GBS) is thought to be a major factor in the virulence of this organism. Transposon mutagenesis was used to obtain isogenic strains of a GBS serotype III clinical isolate (COH 31r/s) with site-specific mutations in the gene(s) responsible for capsule production. The self-conjugative transposon Tn916 was transferred to strain COH 31r/s during incubation with Streptococcus faecalis strain CG110 on membrane filters. Eleven transconjugant clones did not bind type III GBS antiserum by immunoblot. Immunofluorescence, competitive ELISA, and electron microscopy confirmed the absence of detectable GBS type III capsular polysaccharide in one of the transconjugants, COH 31-15. Southern hybridization analysis with a Tn916 probe confirmed the presence of the transposon sequence within each mutant. A 3.0-kilobase EcoRI fragment that flanked the Tn916 sequence was subcloned from mutant COH 31-15. This fragment shared homology with DNA from the other GBS serotypes, suggesting a common sequence for capsulation shared by organisms of different capsular types. Loss of capsule expression resulted in loss of virulence in a neonatal rat model. We conclude that a gene common to all capsular types of GBS is required for surface expression of the type III capsule and that inactivation of this gene by Tn916 results in the loss of virulence.

Downregulation of Forkhead box F1 gene expression in the pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia.

PubMed

Zimmer, J; Takahashi, T; Hofmann, A D; Puri, Prem

2016-12-01

High mortality and morbidity in infants born with congenital diaphragmatic hernia (CDH) are attributed to pulmonary hypoplasia and pulmonary hypertension (PH). Forkhead box (Fox) transcription factors are known to be crucial for cell proliferation and homeostasis. FoxF1 is essential for lung morphogenesis, vascular development, and endothelial proliferation. Mutations in FoxF1 and also the Fox family member FoxC2 have been identified in neonates with PH. In human and experimental models of arterial PH, the Fox protein FoxO1 was found to be downregulated. We hypothesized that Fox expression is altered in the lungs of the nitrofen-induced CDH rat model and investigated the expression of FoxF1, FoxC2, and FoxO1. Following ethical approval (Rec 913b), time-pregnant Sprague-Dawley rats received nitrofen or vehicle on gestational day (D9). Fetuses were sacrificed on D21, inspected for CDH and divided into CDH (n = 11) and control group (n = 11). Gene expression of FoxF1, FoxC2, and FoxO1 was evaluated with qRT-PCR. Detected alterations of mRNA levels were subsequently assessed on the protein level by performing western blot analysis and laser scanning confocal microscopy. The relative mRNA level of FoxF1 was significantly downregulated in CDH lungs compared to controls (FoxF1 CDH 1.047 ± 0.108, FoxF1 Ctrl 1.419 ± 0.01, p = 0.014). Relative mRNA levels of FoxC2 and FoxO1 were not found to be altered between the experimental groups (FoxC2 CDH 30.74 ± 8.925, FoxC2 Ctrl 27.408 ± 7.487, p = 0.776; FoxO1 CDH 0.011 ± 0.002, FoxO1 Ctrl 0.011 ± 0.001, p = 0.809). On the protein level, western blotting demonstrated a reduced pulmonary protein expression of FoxF1 in CDH lungs. Confocal microscopy showed a markedly diminished expression of FoxF1 in the pulmonary vasculature of CDH lungs compared to controls. Our study demonstrates a strikingly reduced expression of FoxF1 in the pulmonary vasculature of nitrofen-induced CDH. Altered FoxF1 gene expression during embryogenesis may participate in vascular maldevelopment resulting in PH in this animal model.

Transformation with green fluorescent protein of Trichoderma harzianum 1051, a strain with biocontrol activity against Crinipellis perniciosa, the agent of witches'-broom disease of cocoa.

PubMed

Inglis, Peter W.; Queiroz, Paulo R.; Valadares-Inglis, M. Cléria

1999-04-01

A plasmid vector for fungal expression of an enhanced, red-shifted variant of the Aequoria victoriae green fluorescent protein was constructed by fusion of the EGFP gene to the highly expressed Aspergillus nidulans gpd promoter and the A. nidulans trpC terminator. This construction was introduced by cotransformation, using benomyl selection, into Trichoderma harzianum strain 1051, a strain being evaluated for the biological control of witches'-broom disease of cocoa caused by Crinipellis perniciosa. Epifluorescence microscopy was used to monitor germination and attachment of stable transformant conidia on the surface of C. perniciosa hyphae.

Plasmodium yoelii yoelii 17XNL constitutively expressing GFP throughout the life cycle.

PubMed

Ono, Takeshi; Tadakuma, Takushi; Rodriguez, Ana

2007-03-01

Plasmodium yoelii is a rodent parasite commonly used as a model to study malaria infection. It is the preferred model parasite for liver-stage immunological studies and is also widely used to study hepatocyte, erythrocyte and mosquito infection. We have generated a P. yoelii yoelii 17XNL line that is stably transfected with the green fluorescent protein (gfp) gene. This parasite line constitutively expresses high levels of GFP during the complete parasite life cycle including liver, blood and mosquito stages. These fluorescent parasites can be used in combination with fluorescence activated cell sorting or live microscopy for a wide range of experimental applications.

Multicellular structures developing during maize microspore culture express endosperm and embryo-specific genes and show different embryogenic potentialities.

PubMed

Massonneau, Agnes; Coronado, Maria-José; Audran, Arthur; Bagniewska, Agnieszka; Mòl, Rafal; Testillano, Pilar S; Goralski, Grzegorz; Dumas, Christian; Risueño, Maria-Carmen; Matthys-Rochon, Elisabeth

2005-07-01

During maize pollen embryogenesis, a range of multicellular structures are formed. Using different approaches, the "nature" of these structures has been determined in terms of their embryogenic potential. In situ molecular identification techniques for gene transcripts and products, and a novel cell tracking system indicated the presence of embryogenic (embryo-like structures, ELS) and non-embryogenic (callus-like structures, CLS) structures that occurred for short periods within the cultures. Some multicellular structures with a compact appearance generated embryos. RT-PCR and fluorescence in situ hybridization (FISH) with confocal microscopy techniques using specific gene markers of the endosperm (ZmESR2, ZmAE3) and embryo (LTP2 and ZmOCL1, ZmOCL3) revealed "embryo" and "endosperm" potentialities in these various multicellular structures present in the cultures. The results presented here showed distinct and specific patterns of gene expression. Altogether, the results demonstrate the presence of different molecules on both embryonic and non-embryonic structures. Their possible roles are discussed in the context of a parallel between embryo/endosperm interactions in planta and embryonic and non-embryonic structure interrelations under in vitro conditions.

Investigation of angiogenesis in bioactive 3-dimensional poly(d,l-lactide-co-glycolide)/nano-hydroxyapatite scaffolds by in vivo multiphoton microscopy in murine calvarial critical bone defect.

PubMed

Li, Jian; Xu, Qiang; Teng, Bin; Yu, Chen; Li, Jian; Song, Liang; Lai, Yu-Xiao; Zhang, Jian; Zheng, Wei; Ren, Pei-Gen

2016-09-15

Reconstruction of critical size bone defects remains a major clinical challenge because of poor bone regeneration, which is usually due to poor angiogenesis during repair. Satisfactory vascularization is a prerequisite for the survival of grafts and the integration of new tissue with existing tissue. In this work, we investigated angiogenesis in 3D scaffolds by in vivo multiphoton microscopy during bone formation in a murine calvarial critical bone defect model and evaluated bone regeneration 8weeks post-implantation. The continuous release of bioactive lentiviral vectors (LV-pdgfb) from the scaffolds could be detected for 5days in vitro. In vivo, the released LV-pdgfb transfected adjacent cells and expressed PDGF-BB, facilitating angiogenesis and enhancing bone regeneration. The expression of both pdgfb and the angiogenesis-related genes vWF and VEGFR2 was significantly increased in the pdgfb gene-carrying scaffold (PHp) group. In addition, microCT scanning and histomorphology results proved that there was more new bone ingrowth in the PHp group than in the PLGA/nHA (PH) and control groups. MicroCT parameters, including BMD, BV/TV, Tb.Sp, and Tb.N indicated that there was significantly more new bone formation in the PHp group than in the other groups. With regard to neovascularization, 8weeks post-implantation, blood vessel areas (BVAs) were 9428±944μm(2), 4090±680.3μm(2), and none in the PHp, PH, and control groups, respectively. At each time point, BVAs in the PHp scaffolds were significantly higher than in the PH scaffolds. To our knowledge, this is the first use of multiphoton microscopy in bone tissue-engineering to investigate angiogenesis in scaffolds in vivo. This method represents a valuable tool for investigating neovascularization in bone scaffolds to determine if a certain scaffold is beneficial to neovascularization. We also proved that delivery of the pdgfb gene alone can improve both angiogenesis and bone regeneration Acronyms. Reconstruction of critical size bone defects remains a major clinical challenge because of poor bone regeneration, which is usually due to poor angiogenesis during repair. Satisfactory vascularization is a prerequisite for the survival of grafts and the integration of new tissue with existing tissue. In this work, we investigated angiogenesis in 3D scaffolds by in vivo multiphoton microscopy during bone formation in a murine calvarial critical bone defect model and evaluated bone regeneration 8weeks post-implantation. To verify that pdgfb-expressing vectors carried by the scaffolds can promote angiogenesis in 3D-printed scaffolds in vivo, we monitored angiogenesis within the implants by multiphoton microscopy. To our knowledge, this is the first study to dynamically investigate angiogenesis in bone tissue engineering scaffolds in vivo. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

A novel recombinant pseudorabies virus expressing parvovirus VP2 gene: Immunogenicity and protective efficacy in swine.

PubMed

Chen, Yang; Guo, Wanzhu; Xu, Zhiwen; Yan, Qigui; Luo, Yan; Shi, Qian; Chen, Dishi; Zhu, Ling; Wang, Xiaoyu

2011-06-16

Porcine parvovirus (PPV) VP2 gene has been successfully expressed in many expression systems resulting in self-assembly of virus-like particles (VLPs) with similar morphology to the native capsid. Here, a pseudorabies virus (PRV) system was adopted to express the PPV VP2 gene. A recombinant PRV SA215/VP2 was obtained by homologous recombination between the vector PRV viral DNA and a transfer plasmid. Then recombinant virus was purified with plaque purification, and its identity confirmed by PCR amplification, Western blot and indirect immunofluorescence (IFA) analyses. Electronic microscopy of PRV SA215/VP2 confirmed self-assembly of both pseudorabies virus and VLPs from VP2 protein. Immunization of piglets with recombinant virus elicited PRV-specific and PPV-specific humoral immune responses and provided complete protection against a lethal dose of PRV challenges. Gilts immunized with recombinant viruses induced PPV-specific antibodies, and significantly reduced the mortality rate of (1 of 28) following virulent PPV challenge compared with the control (7 of 31). Furthermore, PPV virus DNA was not detected in the fetuses of recombinant virus immunized gilts. In this study, a recombinant PRV SA215/VP2 virus expressing PPV VP2 protein was constructed using PRV SA215 vector. The safety, immunogenicity, and protective efficacy of the recombinant virus were demonstrated in piglets and primiparous gilts. This recombinant PRV SA215/VP2 represents a suitable candidate for the development of a bivalent vaccine against both PRV and PPV infection.

Effects of normal saline and selenium-enriched hot spring water on experimentally induced rhinosinusitis in rats.

PubMed

Kim, Dong-Hyun; Yeo, Sang Won

2013-01-01

This prospective, randomized, and controlled study examined the effects of normal saline and selenium-enriched hot spring water on experimentally induced rhinosinusitis in rats. The study comprised two control groups (untreated and saline-treated) and three experimental groups of Sprague Dawley rats. The experimental groups received an instillation of lipopolysaccharide (LPS) only, LPS+normal saline (LPS/saline), or LPS+selenium-enriched hot spring water (LPS/selenium). Histopathological changes were identified using hematoxylin-eosin staining. Leakage of exudate was identified using fluorescence microscopy. Microvascular permeability was measured using the Evans blue dye technique. Expression of the Muc5ac gene was measured using reverse transcription-polymerase chain reaction. Mucosal edema and expression of the Muc5ac gene were significantly lower in the LPS/saline group than in the LPS group. Microvascular permeability, mucosal edema, and expression of the Muc5ac gene were significantly lower in the LPS/selenium group than in the LPS group. Mucosal edema was similar in the LPS/selenium group and LPS/saline group, but capillary permeability and Muc5ac expression were lower in the LPS/selenium group. This study shows that normal saline and selenium-enriched hot spring water reduce inflammatory activity and mucus hypersecretion in LPS-induced rhinosinusitis in rats. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

miR-99 inhibits cervical carcinoma cell proliferation by targeting TRIB2.

PubMed

Xin, Jia-Xuan; Yue, Zhen; Zhang, Shuai; Jiang, Zhong-Hua; Wang, Ping-Yu; Li, You-Jie; Pang, Min; Xie, Shu-Yang

2013-10-01

MicroRNAs (miRNAs) have significant roles in cell processes, including proliferation, apoptosis and stress responses. To investigate the involvement of miR-99 in the inhibition of HeLa cell proliferation, an miR-99 gene expression vector (pU6.1/miR-99), which overexpressed miR-99 in HeLa cells after transient transfection, was constructed. The expression of miR-99 was detected by qPCR. Cell proliferation and apoptosis were analyzed by cell viability, proliferation and apoptosis assays, as well as by electron microscopy. The results showed that overexpression of miR-99 in HeLa cells increased the HeLa cell mortality rate. Moreover, miR-99 overexpression was able to markedly inhibit HeLa cell proliferation according to the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The cell apoptosis rate was significantly higher in pU6.1/miR-99-treated cells compared with that in the control cultures. Increases in intracellular electron density, as well as the proportion of nuclear plasma, blebbing phenomena and apoptotic bodies were observed in pU6.1/miR-99-treated cells compared with control cultures according to electron microscopy analysis. The Tribbles 2 (TRIB2) 3'-untranslated region was also observed to be targeted by miR-99 and the results further demonstrated that miR-99 was able to negatively regulate TRIB2 expression in HeLa cells The results indicate that miR-99 acts as a tumor suppressor gene in HeLa cells, establishing a theoretical basis for its application in cancer therapeutics.

Comparison of six bone-graft substitutes regarding to cell seeding efficiency, metabolism and growth behaviour of human mesenchymal stem cells (MSC) in vitro.

PubMed

Seebach, Caroline; Schultheiss, Judith; Wilhelm, Kerstin; Frank, Johannes; Henrich, Dirk

2010-07-01

Various synthetic bone-graft substitutes are used commercially as osteoconductive scaffolds in the treatment of bone defects and fractures. The role of bone-graft substitutes is changing from osteoconductive conduits for growth to an delivery system for biologic fracture treatments. Achieving optimal bone regeneration requires biologics (e.g. MSC) and using the correct scaffold incorporated into a local environment for bone regeneration. The need for an unlimited supply with high quality bone-graft substitutes continue to find alternatives for bone replacement surgery. This in vitro study investigates cell seeding efficiency, metabolism, gene expression and growth behaviour of MSC sown on six commercially clinical available bone-graft substitutes in order to define their biological properties: synthetic silicate-substituted porous hydroxyapatite (Actifuse ABX), synthetic alpha-TCP (Biobase), synthetic beta-TCP (Vitoss), synthetic beta-TCP (Chronos), processed human cancellous allograft (Tutoplast) and processed bovines hydroxyapatite ceramic (Cerabone). 250,000 MSC derived from human bone marrow (n=4) were seeded onto the scaffolds, respectively. On days 2, 6 and 10 the adherence of MSC (fluorescence microscopy) and cellular activity (MTT assay) were analysed. Osteogenic gene expression (cbfa-1) was analysed by RT-PCR and scanning electron microscopy was performed. The highest number of adhering cells was found on Tutoplast (e.g. day 6: 110.0+/-24.0 cells/microscopic field; p<0.05) followed by Chronos (47.5+/-19.5, p<0.05), Actifuse ABX (19.1+/-4.4), Biobase (15.7+/-9.9), Vitoss (8.8+/-8.7) and Cerabone (8.1+/-2.2). MSC seeded onto Tutoplast showed highest metabolic activity and gene expression of cbfa-1. These data are confirmed by scanning electron microscopy. The cell shapes varied from round-shaped cells to wide spread cells and cell clusters, depending on the bone-graft substitutes. Processed human cancellous allograft is a well-structured and biocompatible scaffold for ingrowing MSC in vitro. Of all other synthetical scaffolds, beta-tricalcium phosphate (Chronos) have shown the best growth behaviour for MSC. Our results indicate that various bone-graft substitutes influence cell seeding efficiency, metabolic activity and growth behaviour of MSC in different manners. We detected a high variety of cellular integration of MSC in vitro, which may be important for bony integration in the clinical setting. 2010 Elsevier Ltd. All rights reserved.

EpsA is an essential gene in exopolysaccharide production in Lactobacillus johnsonii FI9785.

PubMed

Dertli, Enes; Mayer, Melinda J; Colquhoun, Ian J; Narbad, Arjan

2016-07-01

Lactobacillus johnsonii FI9785 has an eps gene cluster which is required for the biosynthesis of homopolymeric exopolysaccharides (EPS)-1 and heteropolymeric EPS-2 as a capsular layer. The first gene of the cluster, epsA, is the putative transcriptional regulator. In this study we showed the crucial role of epsA in EPS biosynthesis by demonstrating that deletion of epsA resulted in complete loss of both EPS-1 and EPS-2 on the cell surface. Plasmid complementation of the epsA gene fully restored EPS production, as confirmed by transmission electron microscopy and nuclear magnetic resonance (NMR) analysis. Furthermore, this complementation resulted in a twofold increase in the expression levels of this gene, which almost doubled amounts of EPS production in comparison with the wild-type strain. Analysis of EPS by NMR showed an increased ratio of the heteropolysaccharide to homopolysaccharide in the complemented strain and allowed identification of the acetylated residue in EPS-2 as the (1,4)-linked βGlcp unit, with the acetyl group located at O-6. These findings indicate that epsA is a positive regulator of EPS production and that EPS production can be manipulated by altering its expression. © 2015 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Application of the laser capture microdissection technique for molecular definition of skeletal cell differentiation in vivo.

PubMed

Benayahu, Dafna; Socher, Rina; Shur, Irena

2008-01-01

Laser capture microdissection (LCM) method allows selection of individual or clustered cells from intact tissues. This technology enables one to pick cells from tissues that are difficult to study individually, sort the anatomical complexity of these tissues, and make the cells available for molecular analyses. Following the cells' extraction, the nucleic acids and proteins can be isolated and used for multiple applications that provide an opportunity to uncover the molecular control of cellular fate in the natural microenvironment. Utilization of LCM for the molecular analysis of cells from skeletal tissues will enable one to study differential patterns of gene expression in the native intact skeletal tissue with reliable interpretation of function for known genes as well as to discover novel genes. Variability between samples may be caused either by differences in the tissue samples (different areas isolated from the same section) or some variances in sample handling. LCM is a multi-task technology that combines histology, microscopy work, and dedicated molecular biology. The LCM application will provide results that will pave the way toward high throughput profiling of tissue-specific gene expression using Gene Chip arrays. Detailed description of in vivo molecular pathways will make it possible to elaborate on control systems to apply for the repair of genetic or metabolic diseases of skeletal tissues.

Biofilm formation by the periodontopathic bacteria Treponema denticola and Porphyromonas gingivalis.

PubMed

Kuramitsu, Howard K; Chen, Wen; Ikegami, Aki

2005-11-01

Periodontitis develops as a result of the interaction of the host with subgingival plaque bacteria. Both Porphyromonas gingivalis and Treponema denticola are frequently associated together in these oral biofilms. The molecular basis for in vitro biofilm formation was investigated for P. gingivalis 381, T. denticola 35405, and mixtures of the two organisms using microtiter plate assays. In addition, the biofilms were examined following confocal laser scanning microscopy. P. gingivalis 381, but not T. denticola strains, formed biofilms in vitro. This property was dependent, in part, on the strain 381 fimA, ppk, and usp genes. Microarray and Northern blot analyses suggested that the expression of the ppk gene was required for maximal expression of the uspA gene. P. gingivalis 381 formed synergistic biofilms when incubated with T. denticola strains. This process was dependent upon the strain 381 rgpB and fimA genes as well as the T. denticola flgE and cfpA genes. P. gingivalis 381 formed synergistic biofilms with T. denticola 35405. These results may be relevant to the previous observations that the two organisms are frequently observed together in subgingival plaque with the spirochetes localized to the exterior of the oral biofilms. It is suggested that other such synergistic effects may also occur between other plaque bacteria.

Biofilm Formation by the Periodontopathic Bacteria Treponema denticola and Porphyromonas gingivalis.

PubMed

Kuramitsu, Howard K; Chen, Wen; Ikegami, Aki

2005-11-01

Periodontitis develops as a result of the interaction of the host with subgingival plaque bacteria. Both Porphyromonas gingivalis and Treponema denticola are frequently associated together in these oral biofilms. The molecular basis for in vitro biofilm formation was investigated for P. gingivalis 381, T. denticola 35405, and mixtures of the two organisms using microtiter plate assays. In addition, the biofilms were examined following confocal laser scanning microscopy. P. gingivalis 381, but not T. denticola strains, formed biofilms in vitro. This property was dependent, in part, on the strain 381 fimA, ppk, and usp genes. Microarray and Northern blot analyses suggested that the expression of the ppk gene was required for maximal expression of the uspA gene. P. gingivalis 381 formed synergistic biofilms when incubated with T. denticola strains. This process was dependent upon the strain 381 rgpB and fimA genes as well as the T. denticola flgE and cfpA genes. P. gingivalis 381 formed synergistic biofilms with T. denticola 35405. These results may be relevant to the previous observations that the two organisms are frequently observed together in subgingival plaque with the spirochetes localized to the exterior of the oral biofilms. It is suggested that other such synergistic effects may also occur between other plaque bacteria. © 2005 American Academy of Periodontology.

Response of sweet orange (Citrus sinensis) to 'Candidatus Liberibacter asiaticus' infection: microscopy and microarray analyses.

PubMed

Kim, Jeong-Soon; Sagaram, Uma Shankar; Burns, Jacqueline K; Li, Jian-Liang; Wang, Nian

2009-01-01

Citrus greening or huanglongbing (HLB) is a devastating disease of citrus. HLB is associated with the phloem-limited fastidious prokaryotic alpha-proteobacterium 'Candidatus Liberibacter spp.' In this report, we used sweet orange (Citrus sinensis) leaf tissue infected with 'Ca. Liberibacter asiaticus' and compared this with healthy controls. Investigation of the host response was examined with citrus microarray hybridization based on 33,879 expressed sequence tag sequences from several citrus species and hybrids. The microarray analysis indicated that HLB infection significantly affected expression of 624 genes whose encoded proteins were categorized according to function. The categories included genes associated with sugar metabolism, plant defense, phytohormone, and cell wall metabolism, as well as 14 other gene categories. The anatomical analyses indicated that HLB bacterium infection caused phloem disruption, sucrose accumulation, and plugged sieve pores. The up-regulation of three key starch biosynthetic genes including ADP-glucose pyrophosphorylase, starch synthase, granule-bound starch synthase and starch debranching enzyme likely contributed to accumulation of starch in HLB-affected leaves. The HLB-associated phloem blockage resulted from the plugged sieve pores rather than the HLB bacterial aggregates since 'Ca. Liberibacter asiaticus' does not form aggregate in citrus. The up-regulation of pp2 gene is related to callose deposition to plug the sieve pores in HLB-affected plants.

The Presence or Absence of Intestinal Microbiota Affects Lipid Deposition and Related Genes Expression in Zebrafish (Danio rerio).

PubMed

Sheng, Yi; Ren, Hui; Limbu, Samwel M; Sun, Yuhong; Qiao, Fang; Zhai, Wanying; Du, Zhen-Yu; Zhang, Meiling

2018-01-01

Understanding how intestinal microbiota alters energy homeostasis and lipid metabolism is a critical process in energy balance and health. However, the exact role of intestinal microbiota in the regulation of lipid metabolism in fish remains unclear. Here, we used two zebrafish models (germ-free and antibiotics-treated zebrafish) to identify the role of intestinal microbiota in lipid metabolism. Conventional and germ-free zebrafish larvae were fed with egg yolk. Transmission electron microscopy was used to detect the presence of lipid droplets in the intestinal epithelium. The results showed that, microbiota increased lipid accumulation in the intestinal epithelium. The mRNA sequencing technology was used to assess genes expression level. We found majority of the differentially expressed genes were related to lipid metabolism. Due to the limitation of germ-free zebrafish larvae, antibiotics-treated zebrafish were also used to identify the relationship between the gut microbiota and the host lipid metabolism. Oil-red staining showed antibiotics-treated zebrafish had less intestinal lipid accumulation than control group. The mRNA expression of genes related to lipid metabolism in liver and intestine was also quantified by using real-time PCR. The results indicated that apoa4 , hsl , cox15 , slc2a1a , and lss were more related to intestinal bacteria in fish, while the influence of intestinal microbiota on the activity of fabp6 , acsl5 , cd36 , and gpat2 was different between the liver and intestine. This study identified several genes regulated by intestinal microbiota. Furthermore, the advantages and disadvantages of each model have been discussed. This study provides valuable information for exploring host-microbiota interactions in zebrafish in future.

Suppression of NGB and NAB/ERabp1 in tomato modifies root responses to potato cyst nematode infestation.

PubMed

Dąbrowska-Bronk, Joanna; Czarny, Magdalena; Wiśniewska, Anita; Fudali, Sylwia; Baranowski, Łukasz; Sobczak, Mirosław; Święcicka, Magdalena; Matuszkiewicz, Mateusz; Brzyżek, Grzegorz; Wroblewski, Tadeusz; Dobosz, Renata; Bartoszewski, Grzegorz; Filipecki, Marcin

2015-05-01

Plant-parasitic nematodes cause significant damage to major crops throughout the world. The small number of genes conferring natural plant resistance and the limitations of chemical control require the development of new protective strategies. RNA interference or the inducible over-expression of nematicidal genes provides an environment-friendly approach to this problem. Candidate genes include NGB, which encodes a small GTP-binding protein, and NAB/ERabp1, which encodes an auxin-binding protein, which were identified as being up-regulated in tomato roots in a transcriptome screen of potato cyst nematode (Globodera rostochiensis) feeding sites. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization confirmed the localized up-regulation of these genes in syncytia and surrounding cells following nematode infection. Gene-silencing constructs were introduced into tomato, resulting in a 20%-98% decrease in transcription levels. Nematode infection tests conducted on transgenic plants showed 57%-82% reduction in the number of G. rostochiensis females in vitro and 30%-46% reduction in pot trials. Transmission electron microscopy revealed a deterioration of cytoplasm, and degraded mitochondria and plastids, in syncytia induced in plants with reduced NAB/ERabp1 expression. Cytoplasm in syncytia induced in plants with low NGB expression was strongly electron translucent and contained very few ribosomes; however, mitochondria and plastids remained intact. Functional impairments in syncytial cytoplasm of silenced plants may result from NGB's role in ribosome biogenesis; this was confirmed by localization of yellow fluorescent protein (YFP)-labelled NGB protein in nucleoli and co-repression of NGB in plants with reduced NAB/ERabp1 expression. These results demonstrate that NGB and NAB/ERabp1 play important roles in the development of nematode-induced syncytia. © 2014 BSPP AND JOHN WILEY & SONS LTD.

Transcriptional profile of Paracoccidioides induced by oenothein B, a potential antifungal agent from the Brazilian Cerrado plant Eugenia uniflora

PubMed Central

2013-01-01

Background The compound oenothein B (OenB), which is isolated from the leaves of Eugenia uniflora, a Brazilian Cerrado plant, interferes with Paracoccidioides yeast cell morphology and inhibits 1,3-β-D-glucan synthase (PbFKS1) transcript accumulation, which is involved in cell wall synthesis. In this work we examined the gene expression changes in Paracoccidioides yeast cells following OenB treatment in order to investigate the adaptive cellular responses to drug stress. Results We constructed differential gene expression libraries using Representational Difference Analysis (RDA) of Paracoccidioides yeast cells treated with OenB for 90 and 180 min. Treatment for 90 min resulted in the identification of 463 up-regulated expressed sequences tags (ESTs) and 104 down-regulated ESTs. For the 180 min treatment 301 up-regulated ESTs and 143 down-regulated were identified. Genes involved in the cell wall biosynthesis, such as GLN1, KRE6 and FKS1, were found to be regulated by OenB. Infection experiments in macrophages corroborated the in vitro results. Fluorescence microscopy showed increased levels of chitin in cells treated with OenB. The carbohydrate polymer content of the cell wall of the fungus was also evaluated, and the results corroborated with the transcriptional data. Several other genes, such as those involved in a variety of important cellular processes (i.e., membrane maintenance, stress and virulence) were found to be up-regulated in response to OenB treatment. Conclusions The exposure of Paracoccidioides to OenB resulted in a complex altered gene expression profile. Some of the changes may represent specific adaptive responses to this compound in this important pathogenic fungus. PMID:24119145

Knockdown of Host Antioxidant Defense Genes Enhances the Effect of Glucantime on Intracellular Leishmania braziliensis in Human Macrophages

PubMed Central

Romero, Ibeth; Soares, Maurilio José; Romanha, Alvaro José

2017-01-01

ABSTRACT Leishmaniasis is a neglected tropical disease that affects millions of people worldwide and represents a major public health problem. Information on protein expression patterns and functional roles within the context of Leishmania-infected human monocyte-derived macrophages (MDMs) under drug treatment conditions is essential for understanding the role of these cells in leishmaniasis treatment. We analyzed functional changes in the expression of human MDM genes and proteins during in vitro infection by Leishmania braziliensis and treatment with Glucantime (SbV), using quantitative PCR (qPCR) arrays, Western blotting, confocal microscopy, and small interfering RNA (siRNA) human gene inhibition assays. Comparison of the results from gene transcription and protein expression analyses revealed that glutathione S-transferase π1 (GSTP1), glutamate-cysteine ligase modifier subunit (GCLM), glutathione reductase (GSR), glutathione synthetase (GSS), thioredoxin (TRX), and ATP-binding cassette, subfamily B, member 5 (ABCB5), were strongly upregulated at both the mRNA and protein levels in human MDMs that were infected and treated, compared to the control group. Subcellular localization studies showed a primarily phagolysosomal location for the ABCB5 transporter, indicating that this protein may be involved in the transport of SbV. By inducing a decrease in L. braziliensis intracellular survival in THP-1 macrophages, siRNA silencing of GSTP1, GSS, and ABCB5 resulted in an increased leishmanicidal effect of SbV exposure in vitro. Our results suggest that human MDMs infected with L. braziliensis and treated with SbV express increased levels of genes participating in antioxidant defense, whereas our functional analyses provide evidence for the involvement of human MDMs in drug detoxification. Therefore, we conclude that GSS, GSTP1, and ABCB5 proteins represent potential targets for enhancing the leishmanicidal activity of Glucantime. PMID:28461312

Functional characterization of Bombyx mori nucleopolyhedrovirus mutant lacking late expression factor 9.

PubMed

Zhang, Y; Shi, Y; Yu, H; Li, J; Quan, Y; Shu, T; Nie, Z; Zhang, Y; Yu, W

Baculoviridae is a family of invertebrate viruses with large double-stranded DNA genomes. Proteins encoded by some late expression factor (lef ) genes are involved in the regulation of viral gene expression. Lef-9 is one of four transcription-specific Lefs, which are components of the virus-encoded RNA polymerase, and can initiate and transcribe late and very late genes. As a multifunctional protein encoded by the Bombyx mori nucleopolyhedrovirus (BmNPV), Lef-9 may be involved in the regulation of viral propagation. However, the underlying mechanism remains unclear. To determine the role of lef-9 in baculovirus infection, lef-9-knockout virus (lef-9-KO-Bacmid virus) was constructed using the Red recombination system, and the Bac-to-Bac system was used to prepare lef-9-repaired virus (lef-9-Re-Bacmid virus). The lef-9-KO virus did not produce infectious viruses or show infection activity, while the lef-9-repaired virus recovered both. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of the transcription levels in wild-type-Bacmid, lef-9-KO-Bacmid, and lef-9-Re-Bacmid viruses showed that the lef-9-KO bacmid had little effect on viral genome replication. However, the transcription levels of the early and late viral genes, lef-3, ie-1, vp39, and p10, were significantly lower in BmN cells transfected with lef-9-KO-Bacmids than in the controls. Electron microscopy showed no visible enveloped virions in cells transfected with lef-9-KO-Bacmids, while many mature virions in cells transfected with lef-9-Re-Bacmid and wt-Bacmid were present. Thus, lef-9 was not essential for viral genome replication, but significantly affected viral gene transcription and expression in all periods of cell life cycle.

In vitro studies of Rickettsia-host cell interactions: Confocal laser scanning microscopy of Rickettsia helvetica-infected eukaryotic cell lines.

PubMed

Speck, Stephanie; Kern, Tanja; Aistleitner, Karin; Dilcher, Meik; Dobler, Gerhard; Essbauer, Sandra

2018-02-01

Rickettsia (R.) helvetica is the most prevalent rickettsia found in Ixodes ricinus ticks in Germany. Several studies reported antibodies against R. helvetica up to 12.5% in humans investigated, however, fulminant clinical cases are rare indicating a rather low pathogenicity compared to other rickettsiae. We investigated growth characteristics of R. helvetica isolate AS819 in two different eukaryotic cell lines with focus on ultra-structural changes of host cells during infection determined by confocal laser scanning microscopy. Further investigations included partially sequencing of rickA, sca4 and sca2 genes, which have been reported to encode proteins involved in cell-to-cell spread and virulence in some rickettsiae. R. helvetica grew constantly but slowly in both cell lines used. Confocal laser scanning microscopy revealed that the dissemination of R. helvetica AS819 in both cell lines was rather mediated by cell break-down and bacterial release than cell-to-cell spread. The cytoskeleton of both investigated eukaryotic cell lines was not altered. R. helvetica possesses rickA, but its expression is not sufficient to promote actin-based motility as demonstrated by confocal laser scanning microscopy. Hypothetical Sca2 and Sca4 proteins were deduced from nucleotide gene sequences but the predicted amino acid sequences were disrupted or truncated compared to other rickettsiae most likely resulting in non-functional proteins. Taken together, these results might give a first hint to the underlying causes of the reduced virulence and pathogenicity of R. helvetica.

Pax3 gene expression is not altered during diaphragmatic development in nitrofen-induced congenital diaphragmatic hernia.

PubMed

Gosemann, Jan-Hendrik; Doi, Takashi; Kutasy, Balazs; Friedmacher, Florian; Dingemann, Jens; Puri, Prem

2012-06-01

Malformations of the pleuroperitoneal folds (PPFs) have been identified as the origin of the diaphragmatic defect in congenital diaphragmatic hernia (CDH). Pax3, expressed in muscle precursor cells (MPCs), plays a key role in regulating myogenesis and muscularization in the fetal diaphragm. Pax3 mutant mice display absence of muscular diaphragm. However, the distribution of muscle precursor cells is reported to be normal in the PPF of the nitrofen-CDH model. We designed this study to investigate the hypothesis that Pax3 gene expression is unaltered in the PPF and developing diaphragm in the nitrofen-induced CDH model. Pregnant rats were treated with nitrofen or vehicle on gestational day (D) 9 and sacrificed on D13, D18, and D21. Pleuroperitoneal folds (D13) and developing diaphragms (D18 and D21) were dissected, total RNA was extracted, and real-time quantitative polymerase chain reaction was performed to determine Pax3 messenger RNA levels. Confocal immunofluorescence microscopy was performed to evaluate protein expression/distribution of Pax3. Relative messenger RNA expression levels of Pax3 in PPFs and developing diaphragms were not significantly different in the nitrofen group compared with controls. Intensity of Pax3 immunofluorescence was also not altered in PPFs and developing diaphragms of the nitrofen group compared with controls. Pax3 gene expression is not altered in the PPFs and developing diaphragm of nitrofen-CDH model, suggesting that the diaphragmatic defect is not caused by disturbance of myogenesis and muscularization. Copyright © 2012 Elsevier Inc. All rights reserved.

Chronology of Islet Differentiation Revealed By Temporal Cell Labeling

PubMed Central

Miyatsuka, Takeshi; Li, Zhongmei; German, Michael S.

2009-01-01

OBJECTIVE Neurogenin 3 plays a pivotal role in pancreatic endocrine differentiation. Whereas mouse models expressing reporters such as eGFP or LacZ under the control of the Neurog3 gene enable us to label cells in the pancreatic endocrine lineage, the long half-life of most reporter proteins makes it difficult to distinguish cells actively expressing neurogenin 3 from differentiated cells that have stopped transcribing the gene. RESEARCH DESIGN AND METHODS In order to separate the transient neurogenin 3 –expressing endocrine progenitor cells from the differentiating endocrine cells, we developed a mouse model (Ngn3-Timer) in which DsRed-E5, a fluorescent protein that shifts its emission spectrum from green to red over time, was expressed transgenically from the NEUROG3 locus. RESULTS In the Ngn3-Timer embryos, green-dominant cells could be readily detected by microscopy or flow cytometry and distinguished from green/red double-positive cells. When fluorescent cells were sorted into three different populations by a fluorescence-activated cell sorter, placed in culture, and then reanalyzed by flow cytometry, green-dominant cells converted to green/red double-positive cells within 6 h. The sorted cell populations were then used to determine the temporal patterns of expression for 145 transcriptional regulators in the developing pancreas. CONCLUSIONS The precise temporal resolution of this model defines the narrow window of neurogenin 3 expression in islet progenitor cells and permits sequential analyses of sorted cells as well as the testing of gene regulatory models for the differentiation of pancreatic islet cells. PMID:19478145

AMP-activated Protein Kinase Mediates Apoptosis in Response to Bioenergetic Stress through Activation of the Pro-apoptotic Bcl-2 Homology Domain-3-only Protein BMF*

PubMed Central

Kilbride, Seán M.; Farrelly, Angela M.; Bonner, Caroline; Ward, Manus W.; Nyhan, Kristine C.; Concannon, Caoimhín G.; Wollheim, Claes B.; Byrne, Maria M.; Prehn, Jochen H. M.

2010-01-01

Heterozygous loss-of-function mutations in the hepatocyte nuclear factor 1A (HNF1A) gene result in the pathogenesis of maturity-onset diabetes-of-the-young type 3, (HNF1A-MODY). This disorder is characterized by a primary defect in metabolism-secretion coupling and decreased beta cell mass, attributed to excessive beta cell apoptosis. Here, we investigated the link between energy stress and apoptosis activation following HNF1A inactivation. This study employed single cell fluorescent microscopy, flow cytometry, gene expression analysis, and gene silencing to study the effects of overexpression of dominant-negative (DN)-HNF1A expression on cellular bioenergetics and apoptosis in INS-1 cells. Induction of DN-HNF1A expression led to reduced ATP levels and diminished the bioenergetic response to glucose. This was coupled with activation of the bioenergetic stress sensor AMP-activated protein kinase (AMPK), which preceded the onset of apoptosis. Pharmacological activation of AMPK using aminoimidazole carboxamide ribonucleotide (AICAR) was sufficient to induce apoptosis in naive cells. Conversely, inhibition of AMPK with compound C or AMPKα gene silencing protected against DN-HNF1A-induced apoptosis. Interestingly, AMPK mediated the induction of the pro-apoptotic Bcl-2 homology domain-3-only protein Bmf (Bcl-2-modifying factor). Bmf expression was also elevated in islets of DN-HNF1A transgenic mice. Furthermore, knockdown of Bmf expression in INS-1 cells using siRNA was sufficient to protect against DN-HNF1A-induced apoptosis. Our study suggests that overexpression of DN-HNF1A induces bioenergetic stress and activation of AMPK. This in turn mediates the transcriptional activation of the pro-apoptotic Bcl-2-homology protein BMF, coupling prolonged energy stress to apoptosis activation. PMID:20841353

The effect of hydrophilic titanium surface modification on macrophage inflammatory cytokine gene expression.

PubMed

Hamlet, Stephen; Alfarsi, Mohammed; George, Roy; Ivanovski, Saso

2012-05-01

Chemical modification of microrough titanium dental implants to produce a hydrophilic surface with increased wettability and improved surface energy has been demonstrated clinically to achieve superior bone wound healing and osseointegration compared to that achieved with a microrough titanium surface alone. As the recruitment of the necessary osseoinductive precursors involved in bone wound healing and osseointegration to the wound site is facilitated by the action of cytokines, this study sought to determine the in vitro effect of hydrophilic surface modification on the expression of pro-inflammatory cytokines from adherent macrophages. The surface topography and composition of the titanium surfaces was characterized by scanning electron microscopy and X-ray photoelectron spectroscopy. Macrophage attachment and proliferation was assessed using an MTT assay. The expression of 84 pro-inflammatory cytokines and chemokines by adherent RAW 264.7 cells, a murine leukaemic monocyte cell line, was assessed by PCR array after 24 h culture on either smooth polished, sand-blasted acid-etched (SLA) or hydrophilic-modified SLA (SLActive) titanium surfaces. Following 24 h culture on titanium, surface microroughness activated pro-inflammatory cytokine gene transcription in RAW 264.7 cells. Although there was no significant difference in the degree of cellular attachment or proliferation of RAW 264.7 cells to the different titanium surfaces, by 24 h the hydrophilic surface elicited a gene expression profile with significant down-regulation of the key pro-inflammatory cytokines Tnfα, IL-1α, IL-1β and the chemokine Ccl-2. Down-regulation of the expression of pro-inflammatory cytokine genes may thus modulate the inflammatory response and may facilitate the enhanced bone wound healing and osseointegration observed clinically using implants with a microrough hydrophilic surface. © 2011 John Wiley & Sons A/S.

Insights into the Role of the Berry-Specific Ethylene Responsive Factor VviERF045

PubMed Central

Leida, Carmen; Dal Rì, Antonio; Dalla Costa, Lorenza; Gómez, Maria D.; Pompili, Valerio; Sonego, Paolo; Engelen, Kristof; Masuero, Domenico; Ríos, Gabino; Moser, Claudio

2016-01-01

During grape ripening, numerous transcriptional and metabolic changes are required in order to obtain colored, sweet, and flavored berries. There is evidence that ethylene, together with other signals, plays an important role in triggering the onset of ripening. Here, we report the functional characterization of a berry-specific Ethylene Responsive Factor (ERF), VviERF045, which is induced just before véraison and peaks at ripening. Phylogenetic analysis revealed it is close to the SHINE clade of ERFs, factors involved in the regulation of wax biosynthesis and cuticle morphology. Transgenic grapevines lines overexpressing VviERF045 were obtained, in vitro propagated, phenotypically characterized, and analyzed for the content of specific classes of metabolites. The effect of VviERF045 was correlated with the level of transgene expression, with high-expressing lines showing stunted growth, discolored and smaller leaves, and a lower level of chlorophylls and carotenoids. One line with intermediate expression, L15, was characterized at the transcriptomic level and showed 573 differentially expressed genes compared to wild type plants. Microscopy and gene expression analyses point toward a major role of VviERF045 in epidermis patterning by acting on waxes and cuticle. They also indicate that VviERF045 affects phenolic secondary metabolism and induces a reaction resembling a plant immune response with modulation of receptor like-kinases and pathogen related genes. These results suggest also a possible role of this transcription factor in berry ripening, likely related to changes in epidermis and cuticle of the berry, cell expansion, a decrease in photosynthetic capacity, and the activation of several defense related genes as well as from the phenylpropanoid metabolism. All these processes occur in the berry during ripening. PMID:28018369

Ectopic Expression of BnaC.CP20.1 Results in Premature Tapetal Programmed Cell Death in Arabidopsis.

PubMed

Song, Liping; Zhou, Zhengfu; Tang, Shan; Zhang, Zhiqiang; Xia, Shengqian; Qin, Maomao; Li, Bao; Wen, Jing; Yi, Bin; Shen, Jinxiong; Ma, Chaozhi; Fu, Tingdong; Tu, Jinxing

2016-09-01

Tapetal programmed cell death (PCD) is essential in pollen grain development, and cysteine proteases are ubiquitous enzymes participating in plant PCD. Although the major papain-like cysteine proteases (PLCPs) have been investigated, the exact functions of many PLCPs are still poorly understood in PCD. Here, we identified a PLCP gene, BnaC.CP20.1, which was closely related to XP_013596648.1 from Brassica oleracea. Quantitative real-time PCR analysis revealed that BnaC.CP20.1 expression was down-regulated in male-sterile lines in oilseed rape, suggesting a connection between this gene and male sterility. BnaC.CP20.1 is especially active in the tapetum and microspores in Brassica napus from the uninucleate stage until formation of mature pollen grains during anther development. On expression of BnaC.CP20.1 prior to the tetrad stage, BnA9::BnaC.CP20.1 transgenic lines in Arabidopsis thaliana showed a male-sterile phenotype with shortened siliques containing fewer or no seeds by self-crossing. Scanning electron microscopy indicated that the reticulate exine was defective in aborted microspores. Callose degradation was delayed and microspores were not released from the tetrad in a timely fashion. Additionally, the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay indicated that BnaC.CP20.1 ectopic expression led to premature tapetal PCD. Transmission electron microscopy analyses further demonstrated that the pollen abortion was due to the absence of tectum connections to the bacula in the transgenic anthers. These findings suggest that timely expression of BnaC.CP20.1 is necessary for tapetal degeneration and pollen wall formation. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Expression and characterization of HPV-16 L1 capsid protein in Pichia pastoris

PubMed Central

Bazan, Silvia Boschi; de Alencar Muniz Chaves, Agtha; Aires, Karina Araújo; Cianciarullo, Aurora Marques; Garcea, Robert L.; Ho, Paulo Lee

2013-01-01

Human papillomaviruses (HPVs) are responsible for the most common human sexually transmitted viral infections. Infection with high-risk HPVs, particularly HPV16, is associated with the development of cervical cancer. The papillomavirus L1 major capsid protein, the basis of the currently marketed vaccines, self-assembles into virus-like particles (VLPs). Here, we describe the expression, purification and characterization of recombinant HPV16 L1 produced by a methylotrophic yeast. A codon-optimized HPV16 L1 gene was cloned into a non-integrative expression vector under the regulation of a methanol-inducible promoter and used to transform competent Pichia pastoris cells. Purification of L1 protein from yeast extracts was performed using heparin–sepharose chromatography, followed by a disassembly/reassembly step. VLPs could be assembled from the purified L1 protein, as demonstrated by electron microscopy. The display of conformational epitopes on the VLPs surface was confirmed by hemagglutination and hemagglutination inhibition assays and by immuno-electron microscopy. This study has implications for the development of an alternative platform for the production of a papillomavirus vaccine that could be provided by public health programs, especially in resource-poor areas, where there is a great demand for low-cost vaccines. PMID:19756360

Inhibitive effects of anti-oxidative vitamins on mannitol-induced apoptosis of vascular endothelial cells.

PubMed

Pan, Kai-yu; Shen, Mei-ping; Ye, Zhi-hong; Dai, Xiao-na; Shang, Shi-qiang

2006-10-01

Study blood vessel injury and gene expression indicating vascular endothelial cell apoptosis induced by mannitol with and without administration of anti-oxidative vitamins. Healthy rabbits were randomly divided into four groups. Mannitol was injected into the vein of the rabbit ear in each animal. Pre-treatment prior to mannitol injection was performed with normal saline (group B), vitamin C (group C) and vitamin E (group D). Blood vessel injury was assessed under electron and light microscopy. In a second experiment, cell culture specimen of human umbilical vein endothelial cells were treated with mannitol. Pre-treatment was done with normal saline (sample B), vitamin C (sample C) and vitamin E (sample D). Total RNA was extracted with the original single step procedure, followed by hybridisation and analysis of gene expression. In the animal experiment, serious blood vessel injury was seen in group A and group B. Group D showed light injury only, and normal tissue without pathological changes was seen in group C. Of all 330 apoptosis-related genes analysed in human cell culture specimen, no significant difference was seen after pre-treatment with normal saline, compared with the gene chip without pre-treatment. On the gene chip pre-treated with vitamin C, 45 apoptosis genes were down-regulated and 34 anti-apoptosis genes were up-regulated. Pre-treatment with vitamin E resulted in the down-regulation of 3 apoptosis genes. Vitamin C can protect vascular endothelial cells from mannitol-induced injury.

Applications of microscopy to genetic therapy of cystic fibrosis and other human diseases.

PubMed

Moninger, Thomas O; Nessler, Randy A; Moore, Kenneth C

2006-01-01

Gene therapy has become an extremely important and active field of biomedical research. Microscopy is an integral component of this effort. This chapter presents an overview of imaging techniques used in our facility in support of cystic fibrosis gene therapy research. Instrumentation used in these studies includes light and confocal microscopy, transmission electron microscopy, and scanning electron microscopy. Techniques outlined include negative staining, cryo-electron microscopy, three-dimentional reconstruction, enzyme cytochemistry, immunocytochemistry, and fluorescence imaging.

Effects of sub-minimum inhibitory concentrations of antimicrobial agents on Streptococcus mutans biofilm formation.

PubMed

Dong, Liping; Tong, Zhongchun; Linghu, Dake; Lin, Yuan; Tao, Rui; Liu, Jun; Tian, Yu; Ni, Longxing

2012-05-01

Many studies have demonstrated that sub-minimum inhibitory concentrations (sub-MICs) of antimicrobial agents can inhibit bacterial biofilm formation. However, the mechanisms by which antimicrobial agents at sub-MICs inhibit biofilm formation remain unclear. At present, most studies are focused on Gram-negative bacteria; however, the effects of sub-MICs of antimicrobial agents on Gram-positive bacteria may be more complex. Streptococcus mutans is a major cariogenic bacterium. In this study, the S. mutans growth curve as well as the expression of genes related to S. mutans biofilm formation were evaluated following treatment with 0.5× MIC of chlorhexidine (CHX), tea polyphenols and sodium fluoride (NaF), which are common anticaries agents. The BioFlux system was employed to generate a biofilm under a controlled flow. Morphological changes of the S. mutans biofilm were observed and analysed using field emission scanning electron microscopy and confocal laser scanning microscopy. The results indicated that these three common anticaries agents could significantly upregulate expression of the genes related to S. mutans biofilm formation, and S. mutans exhibited a dense biofilm with an extensive extracellular matrix following treatment with sub-MICs of NaF and CHX. These findings suggest that sub-MICs of anticaries agents favour S. mutans biofilm formation, which might encourage dental caries progression. Copyright © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Dopamine 5 receptor mediates Ang II type 1 receptor degradation via a ubiquitin-proteasome pathway in mice and human cells

PubMed Central

Li, Hewang; Armando, Ines; Yu, Peiying; Escano, Crisanto; Mueller, Susette C.; Asico, Laureano; Pascua, Annabelle; Lu, Quansheng; Wang, Xiaoyan; Villar, Van Anthony M.; Jones, John E.; Wang, Zheng; Periasamy, Ammasi; Lau, Yuen-Sum; Soares-da-Silva, Patricio; Creswell, Karen; Guillemette, Gaétan; Sibley, David R.; Eisner, Gilbert; Felder, Robin A.; Jose, Pedro A.

2008-01-01

Hypertension is a multigenic disorder in which abnormal counterregulation between dopamine and Ang II plays a role. Recent studies suggest that this counterregulation results, at least in part, from regulation of the expression of both the antihypertensive dopamine 5 receptor (D5R) and the prohypertensive Ang II type 1 receptor (AT1R). In this report, we investigated the in vivo and in vitro interaction between these GPCRs. Disruption of the gene encoding D5R in mice increased both blood pressure and AT1R protein expression, and the increase in blood pressure was reversed by AT1R blockade. Activation of D5R increased the degradation of glycosylated AT1R in proteasomes in HEK cells and human renal proximal tubule cells heterologously and endogenously expressing human AT1R and D5R. Confocal microscopy, Förster/fluorescence resonance energy transfer microscopy, and fluorescence lifetime imaging microscopy revealed that activation of D5R initiated ubiquitination of the glycosylated AT1R at the plasma membrane. The regulated degradation of AT1R via a ubiquitin/proteasome pathway by activation of D5R provides what we believe to be a novel mechanism whereby blood pressure can be regulated by the interaction of 2 counterregulatory GPCRs. Our results therefore suggest that treatments for hypertension might be optimized by designing compounds that can target the AT1R and the D5R. PMID:18464932

Autophagy and skeletal muscles in sepsis.

PubMed

Mofarrahi, Mahroo; Sigala, Ioanna; Guo, Yeting; Godin, Richard; Davis, Elaine C; Petrof, Basil; Sandri, Marco; Burelle, Yan; Hussain, Sabah N A

2012-01-01

Mitochondrial injury develops in skeletal muscles during the course of severe sepsis. Autophagy is a protein and organelle recycling pathway which functions to degrade or recycle unnecessary, redundant, or inefficient cellular components. No information is available regarding the degree of sepsis-induced mitochondrial injury and autophagy in the ventilatory and locomotor muscles. This study tests the hypotheses that the locomotor muscles are more prone to sepsis-induced mitochondrial injury, depressed biogenesis and autophagy induction compared with the ventilatory muscles. Adult male C57/Bl6 mice were injected with i.p. phosphate buffered saline (PBS) or E. coli lipopolysaccharide (LPS, 20 mg/kg) and sacrificed 24 h later. The tibialis anterior (TA), soleus (SOLD) and diaphragm (DIA) muscles were quickly excised and examined for mitochondrial morphological injury, Ca(++) retention capacity and biogenesis. Autophagy was detected with electron microscopy, lipidation of Lc3b proteins and by measuring gene expression of several autophagy-related genes. Electron microscopy revealed ultrastructural injuries in the mitochondria of each muscle, however, injuries were more severe in the TA and SOL muscles than they were in the DIA. Gene expressions of nuclear and mitochondrial DNA transcription factors and co-activators (indicators of biogenesis) were significantly depressed in all treated muscles, although to a greater extent in the TA and SOL muscles. Significant autophagosome formation, Lc3b protein lipidation and upregulation of autophagy-related proteins were detected to a greater extent in the TA and SOL muscles and less so in the DIA. Lipidation of Lc3b and the degree of induction of autophagy-related proteins were significantly blunted in mice expressing a muscle-specific IκBα superrepresor. We conclude that locomotor muscles are more prone to sepsis-induced mitochondrial injury, decreased biogenesis and increased autophagy compared with the ventilatory muscles and that autophagy in skeletal muscles during sepsis is regulated in part through the NFκB transcription factor.

Autophagy and Skeletal Muscles in Sepsis

PubMed Central

Mofarrahi, Mahroo; Sigala, Ioanna; Guo, Yeting; Godin, Richard; Davis, Elaine C.; Petrof, Basil; Sandri, Marco

2012-01-01

Background Mitochondrial injury develops in skeletal muscles during the course of severe sepsis. Autophagy is a protein and organelle recycling pathway which functions to degrade or recycle unnecessary, redundant, or inefficient cellular components. No information is available regarding the degree of sepsis-induced mitochondrial injury and autophagy in the ventilatory and locomotor muscles. This study tests the hypotheses that the locomotor muscles are more prone to sepsis-induced mitochondrial injury, depressed biogenesis and autophagy induction compared with the ventilatory muscles. Methodology/Principal Findings Adult male C57/Bl6 mice were injected with i.p. phosphate buffered saline (PBS) or E. coli lipopolysaccharide (LPS, 20 mg/kg) and sacrificed 24 h later. The tibialis anterior (TA), soleus (SOLD) and diaphragm (DIA) muscles were quickly excised and examined for mitochondrial morphological injury, Ca++ retention capacity and biogenesis. Autophagy was detected with electron microscopy, lipidation of Lc3b proteins and by measuring gene expression of several autophagy-related genes. Electron microscopy revealed ultrastructural injuries in the mitochondria of each muscle, however, injuries were more severe in the TA and SOL muscles than they were in the DIA. Gene expressions of nuclear and mitochondrial DNA transcription factors and co-activators (indicators of biogenesis) were significantly depressed in all treated muscles, although to a greater extent in the TA and SOL muscles. Significant autophagosome formation, Lc3b protein lipidation and upregulation of autophagy-related proteins were detected to a greater extent in the TA and SOL muscles and less so in the DIA. Lipidation of Lc3b and the degree of induction of autophagy-related proteins were significantly blunted in mice expressing a muscle-specific IκBα superrepresor. Conclusion/Significance We conclude that locomotor muscles are more prone to sepsis-induced mitochondrial injury, decreased biogenesis and increased autophagy compared with the ventilatory muscles and that autophagy in skeletal muscles during sepsis is regulated in part through the NFκB transcription factor. PMID:23056618

Human MSC gene expression under simulated microgravity (RPM)

NASA Astrophysics Data System (ADS)

Buravkova, Ludmila; Gershovich, Pavel; Grigoriev, Anatoly

It is generally supposed that microgravity cell response is mediated by some structures of actin cytoskeleton that can be implicated in cell mechanosensitivity. Cytoskeletal reorganization in the microgravity environment can affect gene expression, which results in alterations of cell function. However the direct impact of microgravity on expression of some cytoskeletal genes and encoded proteins remains unknown. Multipotential adult mesechymal stromal cells (MSCs) are the early precursors of bone marrow that can be induced to differentiate into bone-like cells as well as to the other mesenchymal tissues. In our previous experiments we revealed cytoskele-ton alterations and reduced human MSCs growth and osteogenesis in simulated microgravity by Random Positioning Machine. The purpose of this study was to determine the impact of low gravity on F-actin organization and gene expression level of α-, β-, γ-actin, vinculin, cofilin, small GTPase RhoA, Rho kinase (ROCK) and protein expression of some adhesion molecules in cultured hMSCs. Fluorescent microscopy have shown that even 30 min of SMG results in rearrangement of F-actin and the lack of stress fibers in cultured hMSCs. Cell number with abnormal F-actin organization was increased after 6 h, 24 h and 48 h of SMG. On the other hand, after 120 hours of SMG cells displayed partial restoration of F-actin fibers in comparison with 24 h and 48 h. Similarly, near the same restoration was seen in F-actin after readaptation for 24 h in 1g environment after 24 h of SMG. However, the observed alterations in F-actin dimensional organization were accompanied by changes in related proteins gene expression. Real-time PCR revealed slight up-regulation of α-actin expression that became more signifi-cant after 48 h of SMG. Down-regulation of γ-actin was observed after 48 hours of exposure in RPM. Moreover the up-regulation of β-tubulin, cofilin and small GTPase RhoA gene expres-sion was also detected after 48 h of SMG. On the contrary, there was no significant difference between SMG and 1g control group after 120 h of exposure, except up regulation of β-tubulin and, firstly appeared down regulation of vinculin. The same results were obtained when hMSCs were exposed to 24 h readaptation after 24 h of SMG, there were no changes in expression level of all genes of interest. Thus our study has demonstrated that prolonged exposure (more than 120 h) to SMG leads to restoration of hMSC actin cytoskeleton organization. The transient changes in expression level of some genes associated with actin cytoskeleton are supposed to be one of the possible mechanisms which can contribute to first stage of precursor's cellular adaptation to microgravity.

Using HSV-TK/GCV suicide gene therapy to inhibit lens epithelial cell proliferation for treatment of posterior capsular opacification

PubMed Central

Jiang, Yong-Xiang; Liu, Tian-Jing; Yang, Jin; Chen, Yan; Fang, Yan-Wen

2011-01-01

Purpose To establish a novel, targeted lentivirus-based HSV-tk (herpes simplex virus thymidine kinase)/GCV (ganciclovir) gene therapy system to inhibit lens epithelial cell proliferation for treatment of posterior capsular opacification (PCO) after cataract surgery. Methods An enhanced Cre recombinase (Cre/loxP) system with a lentiviral vector expressing Cre under the control of the lens-specific promoter LEP503 (Lenti-LEP503-HSVtk-Cre [LTKCRE]) was constructed, as well as another lentiviral vector containing a switching unit. The latter vector contains a stuffer sequence encoding EGFP (Lenti-hPGK-Loxp-EGFP-pA-Loxp-HSVtk [PGFPTK]) with a functional polyadenylation signal between two loxP sites, followed by the herpes simplex virus thymidine kinase (HSV-tk) gene, both under the control of the human posphoglycerate kinase (hPGK) promoter. Expression of the downstream gene (HSV-tk) is activated by co-expression of Cre. Human lens epithelial cells (HLECs) or retinal pigmental epithelial cells (RPECs) were co-infected with LTKCRE and PGFPTK. The inhibitory effects on HLECs and RPECs infected by the enhanced specific lentiviral vector combination at the concentration of 20 µg/ml GCV were assayed and compared. Results The specific gene expression of Cre and HSV-tk in HLECs is activated by the LEP503 promoter. LTKCRE and PGFPTK co-infected HLECs, but not RPECs, expressed high levels of the HSV-tk protein. After 96 h of GCV treatment, the percentage of apoptotic HLECs infected by the enhanced specific lentiviral vector combination was 87.23%, whereas that of apoptotic RPECs was only 10.12%. Electron microscopy showed that GCV induced apoptosis and necrosis of the infected HLECs. Conclusions The enhanced specific lentiviral vector combination selectively and effectively expressed HSV-tk in HLECs. A concentration of 20 µg/ml, GCV is effective against the proliferation of HLECs in vitro. This cell-type-specific gene therapy using a Cre/loxP lentivirus system may be a feasible treatment strategy to prevent PCO. PMID:21283526

Report of Chinese family with severe dermatitis, multiple allergies and metabolic wasting syndrome caused by novel homozygous desmoglein-1 gene mutation.

PubMed

Cheng, Ruhong; Yan, Ming; Ni, Cheng; Zhang, Jia; Li, Ming; Yao, Zhirong

2016-10-01

Recently, homozygous mutations in the desmoglein-1 (DSG1) gene and heterozygous mutation in the desmoplakin (DSP) gene have been demonstrated to be associated with severe dermatitis, multiple allergies and metabolic wasting (SAM) syndrome (Mendelian Inheritance in Man no. 615508). We aim to identify the molecular basis for a Chinese pedigree of SAM syndrome. A Chinese pedigree of SAM syndrome was subjected to mutation detection in the DSG1 gene. Sequence analysis of the DSG1 gene and quantitative reverse transcriptase polymerase chain reaction analysis for gene expression of DSG1 using cDNA derived from the epidermis of patients and controls were both performed. Skin biopsies were also taken from patients for pathological study and transmission electron microscopy observation. Novel homozygous splicing mutation c.1892-1delG in the exon-intron border of the DSG1 gene has been demonstrated to be associated with SAM syndrome. We report a new family of SAM syndrome of Asian decent and expand the spectrum of mutations in the DSG1 gene. © 2016 Japanese Dermatological Association.

The underlying biological mechanisms of biocompatibility differences between bare and TiN-coated NiTi alloys.

PubMed

Lifeng, Zhao; Yan, Hong; Dayun, Yang; Xiaoying, Lü; Tingfei, Xi; Deyuan, Zhang; Ying, Hong; Jinfeng, Yuan

2011-04-01

TiN coating has been demonstrated to improve the biocompatibility of bare NiTi alloys; however, essential biocompatibility differences between NiTi alloys before and after TiN coating are not known so far. In this study, to explore the underlying biological mechanisms of biocompatibility differences between them, the changes of bare and TiN-coated NiTi alloys in surface chemical composition, morphology, hydrophilicity, Ni ions release, cytotoxicity, apoptosis, and gene expression profiles were compared using energy-dispersive spectroscopy, scanning electron microscopy, contact angle, surface energy, Ni ions release analysis, the methylthiazoltetrazolium (MTT) method, flow cytometry and microarray methods, respectively. Pathways binding to networks and real-time polymerase chain reaction (PCR) were employed to analyze and validate the microarray data, respectively. It was found that, compared with the bare NiTi alloys, TiN coating significantly decreased Ni ions content on the surfaces of the NiTi alloys and reduced the release of Ni ions from the alloys, attenuated the inhibition of Ni ions to the expression of genes associated with anti-inflammatory, and also suppressed the promotion of Ni ions to the expression of apoptosis-related genes. Moreover, TiN coating distinctly improved the hydrophilicity and uniformity of the surfaces of the NiTi alloys, and contributed to the expression of genes participating in cell adhesion and other physiological activities. These results indicate that the TiN-coated NiTi alloys will help overcome the shortcomings of NiTi alloys used in clinical application currently, and can be expected to be a replacement of biomaterials for a medical device field.

Effects of ß-TCP scaffolds on neurogenic and osteogenic differentiation of human embryonic stem cells.

PubMed

Arpornmaeklong, Premjit; Pressler, Michael J

2018-01-01

Extracellular matrix (ECM) and adhesion molecules play crucial roles in regulating growth and differentiation of stem cells. The current study aimed to investigate the effects of beta-tricalcium phosphate (ß-TCP) scaffolds on differentiation and expression of ECM and adhesion molecules of human embryonic stem cells (hESCs). Undifferentiated hESCs were seeded on ß-TCP scaffolds and cell culture plates and cultured in growth and osteogenic medium for 21 days. Scanning electron microscopy (SEM) displayed adhesion and growth of hESCs on the porous ß-TCP scaffolds. Histological analysis, immunohistochemical staining and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) demonstrated that the scaffolds supported growth and differentiation of hESCs. Expression levels of neural crest related genes (AP2a, FoxD3, HNK1, P75, Sox1, Sox10) and osteoblast-related genes (Runx2, SPP1 and BGLA) on the scaffolds in osteogenic medium were significantly higher than on the scaffolds in growth and cell culture plates in osteogenic medium, respectively (p<0.05). Polymerase chain reaction array experiments demonstrated increased expression of ECM and adhesion molecule-related genes on the scaffolds. In conclusion, osteoconductive scaffolds such as ß-TCP scaffolds promoted differentiation of hESCs, particularly expression of genes related to neural crest stem cell and osteoblastic differentiations. Beta-TCP scaffolds could be an alternative cell culture substrate for neural crest and osteogenic differentiation of hESCs. Optimization of culture medium may be necessary to enhance lineage restriction of hESCs on the ß-TCP scaffolds. Copyright © 2017 Elsevier GmbH. All rights reserved.

Effect of Sirtuin-1 on Synaptic Plasticity in Nucleus Accumbens in a Rat Model of Heroin Addiction.

PubMed

Xia, Baijuan; Li, Yixin; Li, Rongrong; Yin, Dan; Chen, Xingqiang; Li, Jie; Liang, Wenmei

2018-06-05

BACKGROUND Synaptic plasticity plays an important role in the process of addiction. This study investigated the relationship between synaptic plasticity and changes in addictive behavior and examined the expression of synaptic plasticity-associated proteins and genes in the nucleus accumbens (NAc) region in different rat models. MATERIAL AND METHODS Heroin addiction, SIRT1-overexpression, and SIRT1-silenced rat models were established. Polymerase chain reaction gene chip technology, immunohistochemistry, Western blotting, and transmission electron microscopy were used to detect changes in synaptic plasticity-related gene and protein expression, and changes in the ultrastructure of synapses, in the NAc. RESULTS Naloxone withdrawal symptoms appeared in the SIRT1-overexpression group. In the SIRT1-silenced group the symptoms were reduced. Immunohistochemistry and Western blotting results showed that FOXO1 expression decreased in the heroin addiction (HA) group but increased in the SIRT1-silenced group (p<0.05). The expression of Cdk5, Nf-κB, PSD95, and Syn was enhanced in the HA group (p<0.05) and further increased in the SIRT1-overexpression group but were reduced in the SIRT1-silenced group (p<0.05). The number of synapses increased in the HA group (p<0.05) along with mitochondrial swelling in the presynaptic membrane and obscuring of the synaptic cleft. CONCLUSIONS SIRT1 and other synaptic plasticity-related genes in NAc are involved in the regulation of heroin addiction. SIRT1 overexpression can increase behavioral sensitization in the NAc of rats, and SIRT1 silencing might ease withdrawal symptoms and reduce conditioned place preferences.

Development of Useful Recombinant Promoter and Its Expression Analysis in Different Plant Cells Using Confocal Laser Scanning Microscopy

PubMed Central

Kumar, Deepak; Sahoo, Dipak K.; Maiti, Indu B.; Dey, Nrisingha

2011-01-01

Background Designing functionally efficient recombinant promoters having reduced sequence homology and enhanced promoter activity will be an important step toward successful stacking or pyramiding of genes in a plant cell for developing transgenic plants expressing desired traits(s). Also basic knowledge regarding plant cell specific expression of a transgene under control of a promoter is crucial to assess the promoter's efficacy. Methodology/Principal Findings We have constructed a set of 10 recombinant promoters incorporating different up-stream activation sequences (UAS) of Mirabilis mosaic virus sub-genomic transcript (MS8, -306 to +27) and TATA containing core domains of Figwort mosaic virus sub-genomic transcript promoter (FS3, −271 to +31). Efficacies of recombinant promoters coupled to GUS and GFP reporter genes were tested in tobacco protoplasts. Among these, a 369-bp long hybrid sub-genomic transcript promoter (MSgt-FSgt) showed the highest activity in both transient and transgenic systems. In a transient system, MSgt-FSgt was 10.31, 2.86 and 2.18 times more active compared to the CaMV35S, MS8 and FS3 promoters, respectively. In transgenic tobacco (Nicotiana tabaccum, var. Samsun NN) and Arabidopsis plants, the MSgt-FSgt hybrid promoter showed 14.22 and 7.16 times stronger activity compared to CaMV35S promoter respectively. The correlation between GUS activity and uidA-mRNA levels in transgenic tobacco plants were identified by qRT-PCR. Both CaMV35S and MSgt-FSgt promoters caused gene silencing but the degree of silencing are less in the case of the MSgt-FSgt promoter compared to CaMV35S. Quantification of GUS activity in individual plant cells driven by the MSgt-FSgt and the CaMV35S promoter were estimated using confocal laser scanning microscopy and compared. Conclusion and Significance We propose strong recombinant promoter MSgt-FSgt, developed in this study, could be very useful for high-level constitutive expression of transgenes in a wide variety of plant cells. PMID:21931783

Hsf-1 affects podocyte markers NPHS1, NPHS2 and WT1 in a transgenic mouse model of TTRVal30Met-related amyloidosis.

PubMed

Petrakis, Ioannis; Mavroeidi, Vasiliki; Stylianou, Kostas; Andronikidi, Eva; Lioudaki, Eirini; Perakis, Kostas; Stratigis, Spyridon; Vardaki, Eleftheria; Zafeiri, Maria; Giannakakis, Kostantinos; Plaitakis, Andreas; Amoiridis, George; Saraiva, Maria Joao; Daphnis, Eugene

2013-09-01

Familial amyloid polyneuropathy is characterized by transthyretin (TTR) deposition in various tissues, including the kidneys. While deposition induces organ dysfunction, renal involvement in TTR-related amyloidosis could manifest from proteinuria to end-stage kidney failure. As proteinuria is considered result of glomerular filtration barrier injury we investigated whether TTR deposition affects either glomerular basement membrane (GBM) or podocytes. Immunohistochemistry, immunoblot and gene expression studies for nephrin, podocin and WT1 were run on renal tissue from human-TTRV30M transgenic mice hemizygous or homozygous for heat shock factor one (Hsf-1). Transmission electron microscopy was used for evaluation of podocyte foot process width (PFW) and GBM thickness in Hsf-1 hemizygous mice with or without TTRV30M or amyloid deposition. Glomeruli of hsf-1 hemizygous transgenic mice showed lower nephrin and podocin protein levels but an increased podocyte number when compared to Hsf-1 homozygous transgenic mice. Nephrin, podocin and WT1 gene expression levels were unaffected by the Hsf-1 carrier status. TTRV30M deposition was associated with increased PFW and GBM thickness. Under the effect of Hsf-1 hemizygosity, TTRV30M deposition has deleterious effects on GBM thickness, PFW and slit diaphragm composition, without affecting nephrin and podocin gene expression.

Antiangiogenic Effects and Therapeutic Targets of Azadirachta indica Leaf Extract in Endothelial Cells

PubMed Central

Mahapatra, Saswati; Young, Charles Y. F.; Kohli, Manish; Karnes, R. Jeffrey; Klee, Eric W.; Holmes, Michael W.; Tindall, Donald J.; Donkena, Krishna Vanaja

2012-01-01

Azadirachta indica (common name: neem) leaves have been found to possess immunomodulatory, anti-inflammatory and anti-carcinogenic properties. The present study evaluates anti-angiogenic potential of ethanol extract of neem leaves (EENL) in human umbilical vein endothelial cells (HUVECs). Treatment of HUVECs with EENL inhibited VEGF induced angiogenic response in vitro and in vivo. The in vitro proliferation, invasion and migration of HUVECs were suppressed with EENL. Nuclear fragmentation and abnormally small mitochondria with dilated cristae were observed in EENL treated HUVECs by transmission electron microscopy. Genome-wide mRNA expression profiling after treatment with EENL revealed differentially regulated genes. Expression changes of the genes were validated by quantitative real-time polymerase chain reaction. Additionally, increase in the expression of HMOX1, ATF3 and EGR1 proteins were determined by immunoblotting. Analysis of the compounds in the EENL by mass spectrometry suggests the presence of nimbolide, 2′,3′-dehydrosalannol, 6-desacetyl nimbinene and nimolinone. We further confirmed antiproliferative activity of nimbolide and 2′,3′-dehydrosalannol in HUVECs. Our results suggest that EENL by regulating the genes involved in cellular development and cell death functions could control cell proliferation, attenuate the stimulatory effects of VEGF and exert antiangiogenic effects. EENL treatment could have a potential therapeutic role during cancer progression. PMID:22461839

Sentan: A Novel Specific Component of the Apical Structure of Vertebrate Motile Cilia

PubMed Central

Yuba-Kubo, Akiko; Tsukita, Sachiko; Tsukita, Shoichiro; Amagai, Masayuki

2008-01-01

Human respiratory and oviductal cilia have specific apical structures characterized by a narrowed distal portion and a ciliary crown. These structures are conserved among vertebrates that have air respiration systems; however, the molecular components of these structures have not been defined, and their functions are unknown. To identify the molecular component(s) of the cilia apical structure, we screened EST libraries to identify gene(s) that are exclusively expressed in ciliated tissues, are transcriptionally up-regulated during in vitro ciliogenesis, and are not expressed in testis (because sperm flagella have no such apical structures). One of the identified gene products, named sentan, was localized to the distal tip region of motile cilia. Using anti-sentan polyclonal antibodies and electron microscopy, sentan was shown to localize exclusively to the bridging structure between the cell membrane and peripheral singlet microtubules, which specifically exists in the narrowed distal portion of cilia. Exogenously expressed sentan showed affinity for the membrane protrusions, and a protein–lipid binding assay revealed that sentan bound to phosphatidylserine. These findings suggest that sentan is the first molecular component of the ciliary tip to bridge the cell membrane and peripheral singlet microtubules, making the distal portion of the cilia narrow and stiff to allow for better airway clearance or ovum transport. PMID:18829862

Smad4 deletion in blood vessel endothelial cells promotes ovarian cancer metastasis.

PubMed

Yang, Jie; Wang, Ya; Zeng, Zhen; Qiao, Long; Zhuang, Liang; Gao, Qinglei; Ma, Ding; Huang, Xiaoyuan

2017-05-01

SMAD4 is a critical co-smad in signal transduction pathways activated in response to transforming growth factor-β (TGF-β)-related ligands, regulating cell growth and differentiation. The roles played by SMAD4 inactivation in tumors highlighted it as a tumor-suppressor gene. Herein, we report that loss of SMAD4 expression in vascular endothelial cells promotes ovarian cancer invasion. SiRNA transfer of this gene in the HUVEC reduced SMAD4 protein expression and function. Although it reduced the vessel endothelial cell tubule formation in vitro and in vivo, it did not affect the tumor growth significantly in vivo. However, it weakened the barrier integrity in endothelial cells and increased vessel permeability and the ovarian cancer liver metastasis. We documented reduced angiogenesis and increased invasion histologically and by intravital microscopy, and gained mechanistic insight at the messenger and gene level. Finally, we found a negative reciprocal regulation between SMAD4 and FYN. FYN is one of the Src family kinases (SFK), activation of which can cause dissociation of cell-cell junctions and adhesion, resulting in paracellular hypermeability. Upon SMAD4 deletion, we detected high expression levels of FYN in vessel endothelial cells, suggesting the mechanism of the ovarian tumor cells cross the endothelial barrier and transform to an invasive phenotype.

Molecular characterization of a subtilase from the vascular wilt fungus Fusarium oxysporum.

PubMed

Di Pietro, A; Huertas-González, M D; Gutierrez-Corona, J F; Martínez-Cadena, G; Méglecz, E; Roncero, M I

2001-05-01

The gene prt1 was isolated from the tomato vascular wilt fungus Fusarium oxysporum f. sp. lycopersici, whose predicted amino acid sequence shows significant homology with subtilisin-like fungal proteinases. Prt1 is a single-copy gene, and its structure is highly conserved among different formae speciales of F. oxysporum. Prt1 is expressed constitutively at low levels during growth on different carbon and nitrogen sources and strongly induced in medium containing collagen and glucose. As shown by reverse transcription-polymerase chain reaction and fluorescence microscopy of F. oxysporum strains carrying a prt1-promoter-green fluorescent protein fusion, prt1 is expressed at low levels during the entire cycle of infection on tomato plants. F. oxysporum strains transformed with an expression vector containing the prt1 coding region fused to the inducible endopolygalacturonase pg1 gene promoter and grown under promoter-inducing conditions secreted high levels of extracellular subtilase activity that resolved into a single peak of pI 4.0 upon isoelectric focusing. The active fraction produced two clearing bands of 29 and 32 kDa in sodium dodecyl sulfate gels containing gelatin. Targeted inactivation of prt1 in F. oxysporum f. sp. lycopersici had no detectable effect on mycelial growth, sporulation, and pathogenicity on tomato plants.

The LuxS/AI-2 Quorum-Sensing System of Streptococcus pneumoniae Is Required to Cause Disease, and to Regulate Virulence- and Metabolism-Related Genes in a Rat Model of Middle Ear Infection.

PubMed

Yadav, Mukesh K; Vidal, Jorge E; Go, Yoon Y; Kim, Shin H; Chae, Sung-Won; Song, Jae-Jun

2018-01-01

Objective: Streptococcus pneumoniae colonizes the nasopharynx of children, and from nasopharynx it could migrate to the middle ear and causes acute otitis media (AOM). During colonization and AOM, the pneumococcus forms biofilms. In vitro biofilm formation requires a functional LuxS/AI-2 quorum-sensing system. We investigated the role of LuxS/AI-2 signaling in pneumococcal middle ear infection, and identified the genes that are regulated by LuxS/AI-2 during pneumococcal biofilm formation. Methods: Streptococcus pneumoniae D39 wild-type and an isogenic D39Δ luxS strain were utilized to evaluate in vitro biofilm formation, and in vivo colonization and epithelial damage using a microtiter plate assay and a rat model of pneumococcal middle ear infection, respectively. Biofilm structures and colonization and epithelial damage were evaluated at the ultrastructural level by scanning electron microscopy and confocal microscopy. Microarrays were used to investigate the global genes that were regulated by LuxS/AI-2 during biofilm formation. Results: The biofilm biomass and density of D39Δ luxS were significantly ( p < 0.05) lower than those of D39 wild-type. SEM and confocal microscopy revealed that D39Δ luxS formed thin biofilms in vitro compared with D39 wild-type. The in vivo model of middle ear infection showed that D39Δ luxS resulted in ~60% less ( p < 0.05) bacterial colonization than the wild-type. SEM analysis of the rat middle ears revealed dense biofilm-like cell debris deposited on the cilia in wild-type D39-infected rats. However, little cell debris was deposited in the middle ears of the D39Δ luxS -inoculated rats, and the cilia were visible. cDNA-microarray analysis revealed 117 differentially expressed genes in D39Δ luxS compared with D39 wild-type. Among the 66 genes encoding putative proteins and previously characterized proteins, 60 were significantly downregulated, whereas 6 were upregulated. Functional annotation revealed that genes involved in DNA replication and repair, ATP synthesis, capsule biosynthesis, cell division, the cell cycle, signal transduction, transcription regulation, competence, virulence, and carbohydrate metabolism were downregulated in the absence of LuxS/AI-2. Conclusion: The S. pneumoniae LuxS/AI-2 quorum-sensing system is necessary for biofilm formation and the colonization of the ear epithelium, and caused middle ear infection in the rat model. LuxS/AI-2 regulates the expression of the genes involved in virulence and bacterial fitness during pneumococcal biofilm formation.

A novel fluorescent sensor for measurement of CFTR function by flow cytometry.

PubMed

Vijftigschild, Lodewijk A W; van der Ent, Cornelis K; Beekman, Jeffrey M

2013-06-01

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis. CFTR-dependent iodide transport measured by fluorescent quenching of ectopically expressed halide-sensitive yellow fluorescent protein (YFP) is widely being used to study CFTR function by microscopy or plate readers. Since YFP fluorescence in these systems is dependent on YFP expression levels and iodide concentration, differences in sensor expression level between experimental units are normalized at the start of each experiment. To allow accurate measurement of CFTR function by flow cytometry, we reasoned that co-expression of an iodide insensitive fluorescent protein would allow for normalization of sensor expression levels and more accurate quantification of CFTR function. Our data indicated that dsRed and mKate fluorescence are iodide insensitive, and we determined an optimal format for co-expression of these fluorescent proteins with halide-sensitive YFP. We showed using microscopy that ratiometric measurement (YFP/mKate) corrects for differences in sensor expression levels. Ratiometric measurements were essential to accurately measure CFTR function by flow cytometry that we here describe for the first time. Mixing of wild type or mutant CFTR expressing cells indicated that addition of approximately 10% of wild type CFTR expressing cells could be distinguished by ratiometric YFP quenching. Flow cytometric ratiometric YFP quenching also allowed us to study CFTR mutants associated with differential residual function upon ectopic expression. Compared with conventional plate-bound CFTR function assays, the flow cytometric approach described here can be used to study CFTR function in suspension cells. It may be further adapted to study CFTR function in heterologous cell populations using cell surface markers and selection of cells that display high CFTR function by cell sorting. Copyright © 2013 International Society for Advancement of Cytometry.

Enhanced biofilm formation and melanin synthesis by the oyster settlement-promoting Shewanella colwelliana is related to hydrophobic surface and simulated intertidal environment.

PubMed

Mitra, Sayani; Gachhui, Ratan; Mukherjee, Joydeep

2015-01-01

A direct relationship between biofilm formation and melanogenesis in Shewanella colwelliana with increased oyster recruitment is already established. Previously, S. colwelliana was grown in a newly patented biofilm-cultivation device, the conico-cylindrical flask (CCF), offering interchangeable hydrophobic/hydrophilic surfaces. Melanization was enhanced when S. colwelliana was cultivated in a hydrophobic vessel compared with a hydrophilic vessel. In the present study, melanogenesis in the CCF was positively correlated with increased architectural parameters of the biofilm (mean thickness and biovolume obtained by confocal laser scanning microscopy) and melanin gene (melA) expression observed by densitometry. Niche intertidal conditions were mimicked in a process operated in an ultra-low-speed rotating disk bioreactor, which demonstrated enhanced biofilm formation, melanogenesis, exopolysaccharide synthesis and melA gene expression compared with a process where 12-h periodic immersion and emersion was prevented. The wettability properties of the settling plane as well as intermittent wetting and drying, which influenced biofilm formation and melA expression, may affect oyster settlement in nature.

Silver nanoparticles induce tight junction disruption and astrocyte neurotoxicity in a rat blood-brain barrier primary triple coculture model.

PubMed

Xu, Liming; Dan, Mo; Shao, Anliang; Cheng, Xiang; Zhang, Cuiping; Yokel, Robert A; Takemura, Taro; Hanagata, Nobutaka; Niwa, Masami; Watanabe, Daisuke

2015-01-01

Silver nanoparticles (Ag-NPs) can enter the brain and induce neurotoxicity. However, the toxicity of Ag-NPs on the blood-brain barrier (BBB) and the underlying mechanism(s) of action on the BBB and the brain are not well understood. To investigate Ag-NP suspension (Ag-NPS)-induced toxicity, a triple coculture BBB model of rat brain microvascular endothelial cells, pericytes, and astrocytes was established. The BBB permeability and tight junction protein expression in response to Ag-NPS, NP-released Ag ions, and polystyrene-NP exposure were investigated. Ultrastructural changes of the microvascular endothelial cells, pericytes, and astrocytes were observed using transmission electron microscopy (TEM). Global gene expression of astrocytes was measured using a DNA microarray. A triple coculture BBB model of primary rat brain microvascular endothelial cells, pericytes, and astrocytes was established, with the transendothelial electrical resistance values >200 Ω·cm(2). After Ag-NPS exposure for 24 hours, the BBB permeability was significantly increased and expression of the tight junction (TJ) protein ZO-1 was decreased. Discontinuous TJs were also observed between microvascular endothelial cells. After Ag-NPS exposure, severe mitochondrial shrinkage, vacuolations, endoplasmic reticulum expansion, and Ag-NPs were observed in astrocytes by TEM. Global gene expression analysis showed that three genes were upregulated and 20 genes were downregulated in astrocytes treated with Ag-NPS. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the 23 genes were associated with metabolic processes, biosynthetic processes, response to stimuli, cell death, the MAPK pathway, and so on. No GO term and KEGG pathways were changed in the released-ion or polystyrene-NP groups. Ag-NPS inhibited the antioxidant defense of the astrocytes by increasing thioredoxin interacting protein, which inhibits the Trx system, and decreasing Nr4a1 and Dusp1. Meanwhile, Ag-NPS induced inflammation and apoptosis through modulation of the MAPK pathway or B-cell lymphoma-2 expression or mTOR activity in astrocytes. These results draw our attention to the importance of Ag-NP-induced toxicity on the neurovascular unit and provide a better understanding of its toxicological mechanisms on astrocytes.

The Activation of the Rat Insulin Gene II by BETA2 and PDX-1 in Rat Insulinoma Cells Is Repressed by Pax6

PubMed Central

Wolf, Gabriele; Hessabi, Behnam; Karkour, Anke; Henrion, Ulrike; Dahlhaus, Meike; Ostmann, Annett; Giese, Bernd; Fraunholz, Martin; Grabarczyk, Piotr; Jack, Robert; Walther, Reinhard

2010-01-01

The transcriptional transactivator Pax6 binds the pancreatic islet cell-specific enhancer sequence (PISCES) of the rat insulin I gene. However the human, mouse, and rat insulin gene II promoters do not contain a PISCES element. To analyze the role of Pax6 in those PISCES-less promoters, we investigated its influence on rat insulin gene II expression and included in our studies the main activators: pancreatic and duodenal homeobox protein-1 (PDX-1) and BETA2/E47. Luciferase assays, Northern blots, and RIA were used to study effects of Pax6 overexpression, gel shift and chromatin precipitation assays to study its binding to the DNA, and yeast two-hybrid assays and glutathione S transferase capture assays to investigate its interactions with PDX-1 and BETA2. Finally, glucose-dependent intracellular transport of Pax6 was demonstrated by fluorescence microscopy. Overexpression of Pax6 prevents activation of the rat insulin II gene by BETA2 and PDX-1 and hence suppresses insulin synthesis and secretion. In vitro, Pax6 binds to the A-boxes, thereby blocking binding of PDX-1, and at the same time, its paired domain interacts with BETA2. Fluorescence microscopy demonstrated that the nuclear-cytoplasmic localization of Pax6 and PDX-1 are oppositely regulated by glucose. From the results, it is suggested that at low concentrations of glucose, Pax6 is localized in the nucleus and prevents the activation of the insulin gene by occupying the PDX-1 binding site and by interacting with BETA2. PMID:20943817

Morphological observation of the stria vascularis in midkine and pleiotrophin knockout mice.

PubMed

Sone, Michihiko; Muramatsu, Hisako; Muramatsu, Takashi; Nakashima, Tsutomu

2011-02-01

Midkine and Pleiotrophin are low molecular weight basic proteins with closely related structures and serve as growth/differentiation factors. They have been reported to be expressed in the cochlea during the embryonic and perinatal periods. In the present study, we focused on the roles of midkine and pleiotrophin in the stria vascularis and investigated morphological changes using mice deficient in these genes. Midkine knockout, pleiotrophin knockout, and double knockout mice were used and compared to wild-type mice. Auditory brain stem responses (ABRs) and cochlear blood flows were measured in each type of mice. Pathological changes in the stria vascularis were examined by light microscopy, including immunohistochemical staining with anti-Kir4.1 antibody, and electron microscopy. Hearing thresholds examined by ABRs were significantly higher in midkine knockout and pleiotrophin knockout mice than in wild-type mice. Double knockout mice showed higher thresholds compared to midkine knockout and pleiotrophin knockout mice. Blood flow in the lateral walls did not significantly differ and light microscopy examination showed an almost normal appearance of the stria vascularis in these knockout mice. However, the expression of Kir4.1 was weak in the knockout mice and severe vacuolar degeneration was observed by electron microscopy in the intermediate cells of the double knockout mice. The present study demonstrates that midkine and pleiotrophin play some roles for the morphological maintenance of intermediate cell in the stria vascularis. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

FvSNF1, the sucrose non-fermenting protein kinase gene of Fusarium virguliforme, is required for cell-wall-degrading enzymes expression and sudden death syndrome development in soybean.

PubMed

Islam, Kazi T; Bond, Jason P; Fakhoury, Ahmad M

2017-08-01

Fusarium virguliforme is a soil-borne pathogenic fungus that causes sudden death syndrome (SDS) in soybean. Its pathogenicity is believed to require the activity of cell-wall-degrading enzymes (CWDEs). The sucrose non-fermenting protein kinase 1 gene (SNF1) is a key component of the glucose de-repression pathway in yeast, and a regulator of gene expression for CWDEs in some plant pathogenic fungi. To elucidate the functional role of the SNF1 homolog in F. virguliforme, FvSNF1 was disrupted using a split-marker strategy. Disruption of FvSNF1 in F. virguliforme abolishes galactose utilization and causes poor growth on xylose, arabinose and sucrose. However, the resulting Fvsnf1 mutant grew similar to wild-type and ectopic transformants on glucose, fructose, maltose, or pectin as the main source of carbon. The Fvsnf1 mutant displayed no expression of the gene-encoding galactose oxidase (GAO), a secretory enzyme that catalyzes oxidation of D-galactose. It also exhibited a significant reduction in the expression of several CWDE-coding genes in contrast to the wild-type strain. Greenhouse pathogenicity assays revealed that the Fvsnf1 mutant was severely impaired in its ability to cause SDS on challenged soybean plants. Microscopy and microtome studies on infected roots showed that the Fvsnf1 mutant was defective in colonizing vascular tissue of infected plants. Cross and longitudinal sections of infected roots stained with fluorescein-labeled wheat germ agglutinin and Congo red showed that the Fvsnf1 mutant failed to colonize the xylem vessels and phloem tissue at later stages of infection. Quantification of the fungal biomass in inoculated roots further confirmed a reduced colonization of roots by the Fvsnf1 mutant when compared to the wild type. These findings suggest that FvSNF1 regulates the expression of CWDEs in F. virguliforme, thus affecting the virulence of the fungus on soybean.

In Vitro Evaluation of the Biological Responses of Canine Macrophages Challenged with PLGA Nanoparticles Containing Monophosphoryl Lipid A.

PubMed

Guldner, Delphine; Hwang, Julianne K; Cardieri, Maria Clara D; Eren, Meaghan; Ziaei, Parissa; Norton, M Grant; Souza, Cleverson D

2016-01-01

Poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) have been considerably studied as a promising biodegradable delivery system to induce effective immune responses and to improve stability, safety, and cost effectiveness of vaccines. The study aimed at evaluating early inflammatory effects and cellular safety of PLGA NPs, co-encapsulating ovalbumin (PLGA/OVA NPs), as a model antigen and the adjuvant monophosphoryl lipid A (PLGA/MPLA NPs) as an adjuvant, on primary canine macrophages. The PLGA NPs constructs were prepared following the emulsion-solvent evaporation technique and further physic-chemically characterized. Peripheral blood mononuclear cells were isolated from canine whole blood by magnetic sorting and further cultured to generate macrophages. The uptake of PLGA NP constructs by macrophages was demonstrated by flow cytometry, transmission electron microscopy and confocal microscopy. Macrophage viability and morphology were evaluated by trypan blue exclusion and light microscopy. Macrophages were immunophenotyped for the expression of MHC-I and MHC-II and gene expression of Interleukin-10 (IL-10), Interleukin-12 (IL-12p40), and tumor necrosis factor alpha (TNF-α) were measured. The results showed that incubation of PLGA NP constructs with macrophages revealed effective early uptake of the PLGA NPs without altering the viability of macrophages. PLGA/OVA/MPLA NPs strongly induced TNF-α and IL-12p40 expression by macrophages as well as increase relative expression of MHC-I but not MHC-II molecules. Taken together, these results indicated that PLGA NPs with addition of MPLA represent a good model, when used as antigen carrier, for further, in vivo, work aiming to evaluate their potential to induce strong, specific, immune responses in dogs.

Frequency domain analysis of noise in simple gene circuits

NASA Astrophysics Data System (ADS)

Cox, Chris D.; McCollum, James M.; Austin, Derek W.; Allen, Michael S.; Dar, Roy D.; Simpson, Michael L.

2006-06-01

Recent advances in single cell methods have spurred progress in quantifying and analyzing stochastic fluctuations, or noise, in genetic networks. Many of these studies have focused on identifying the sources of noise and quantifying its magnitude, and at the same time, paying less attention to the frequency content of the noise. We have developed a frequency domain approach to extract the information contained in the frequency content of the noise. In this article we review our work in this area and extend it to explicitly consider sources of extrinsic and intrinsic noise. First we review applications of the frequency domain approach to several simple circuits, including a constitutively expressed gene, a gene regulated by transitions in its operator state, and a negatively autoregulated gene. We then review our recent experimental study, in which time-lapse microscopy was used to measure noise in the expression of green fluorescent protein in individual cells. The results demonstrate how changes in rate constants within the gene circuit are reflected in the spectral content of the noise in a manner consistent with the predictions derived through frequency domain analysis. The experimental results confirm our earlier theoretical prediction that negative autoregulation not only reduces the magnitude of the noise but shifts its content out to higher frequency. Finally, we develop a frequency domain model of gene expression that explicitly accounts for extrinsic noise at the transcriptional and translational levels. We apply the model to interpret a shift in the autocorrelation function of green fluorescent protein induced by perturbations of the translational process as a shift in the frequency spectrum of extrinsic noise and a decrease in its weighting relative to intrinsic noise.

Gene Expression of Pneumocystis murina after Treatment with Anidulafungin Results in Strong Signals for Sexual Reproduction, Cell Wall Integrity, and Cell Cycle Arrest, Indicating a Requirement for Ascus Formation for Proliferation.

PubMed

Cushion, Melanie T; Ashbaugh, Alan; Hendrix, Keeley; Linke, Michael J; Tisdale, Nikeya; Sayson, Steven G; Porollo, Aleksey

2018-05-01

The echinocandins are a class of antifungal agents that target β-1,3-d-glucan (BG) biosynthesis. In the ascigerous Pneumocystis species, treatment with these drugs depletes the ascus life cycle stage, which contains BG, but large numbers of forms which do not express BG remain in the infected lungs. In the present study, the gene expression profiles of Pneumocystis murina were compared between infected, untreated mice and mice treated with anidulafungin for 2 weeks to understand the metabolism of the persisting forms. Almost 80 genes were significantly up- or downregulated. Like other fungi exposed to echinocandins, genes associated with sexual replication, cell wall integrity, cell cycle arrest, and stress comprised the strongest upregulated signals in P. murina from the treated mice. The upregulation of the P. murina β-1,3-d-glucan endohydrolase and endo-1,3-glucanase was notable and may explain the disappearance of the existing asci in the lungs of treated mice since both enzymes can degrade BG. The biochemical measurement of BG in the lungs of treated mice and fluorescence microscopy with an anti-BG antibody supported the loss of BG. Downregulated signals included genes involved in cell replication, genome stability, and ribosomal biogenesis and function and the Pneumocystis -specific genes encoding the major surface glycoproteins (Msg). These studies suggest that P. murina attempted to undergo sexual replication in response to a stressed environment and was halted in any type of proliferative cycle, likely due to a lack of BG. Asci appear to be a required part of the life cycle stage of Pneumocystis , and BG may be needed to facilitate progression through the life cycle via sexual replication. Copyright © 2018 Cushion et al.

Exosomes play an important role in the process of psoralen reverse multidrug resistance of breast cancer.

PubMed

Wang, Xiaohong; Xu, Chengfeng; Hua, Yitong; Sun, Leitao; Cheng, Kai; Jia, Zhongming; Han, Yong; Dong, Jianli; Cui, Yuzhen; Yang, Zhenlin

2016-12-01

Release of exosomes have been shown to play critical roles in drug resistance by delivering cargo. Targeting the transfer of exosomes from resistant cells to sensitive cells may be an approach to overcome some cases of drug resistance. In this study, we investigated the potential role of exosomes in the process of psoralen reverse multidrug resistance of MCF-7/ADR cells. Exosomes were isolated by differential centrifugation of culture media from MCF-7/ADR cells (ADR/exo) and MCF-7 parental cells (S/exo). Exosomes were characterized by morphology, exosomal markers and size distribution. The ability of ADR/exo to transfer multidrug resistance was assessed by MTT and real-time quantitative PCR. The different formation and secretion of exosomes were detected by immunofluorescence and transmission electron microscopy. Then we performed comparative transcriptomic analysis using RNA-Seq technology and real-time quantitative PCR to better understand the gene expression regulation in exosmes formation and release after psoralen treatment. Our data showed that exosomes derived from MCF-7/ADR cells were able to promote active sequestration of drugs and could induce a drug resistance phenotype by transferring drug-resistance-related gene MDR-1 and P-glycoprotein protein. Psoralen could reduce the formation and secretion of exosomes to overcome drug resistance. There were 21 differentially expressed genes. Gene ontology (GO) pathway analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the most significantly expressed genes were linked to PPAR and P53 signaling pathways which were related to exosomes formation, secretion and cargo sorting. Psoralen can affect the exosomes and induce the reduction of resistance transmission via exosomes might through PPAR and P53 signaling pathways, which might provide a novel strategy for breast cancer resistance to chemotherapy in the future.

Enterococcus faecalis infection causes inflammation, intracellular oxphos-independent ROS production, and DNA damage in human gastric cancer cells.

PubMed

Strickertsson, Jesper A B; Desler, Claus; Martin-Bertelsen, Tomas; Machado, Ana Manuel Dantas; Wadstrøm, Torkel; Winther, Ole; Rasmussen, Lene Juel; Friis-Hansen, Lennart

2013-01-01

Achlorhydria caused by e.g. atrophic gastritis allows for bacterial overgrowth, which induces chronic inflammation and damage to the mucosal cells of infected individuals driving gastric malignancies and cancer. Enterococcus faecalis (E. faecalis) can colonize achlohydric stomachs and we therefore wanted to study the impact of E. faecalis infection on inflammatory response, reactive oxygen species (ROS) formation, mitochondrial respiration, and mitochondrial genetic stability in gastric mucosal cells. To separate the changes induced by bacteria from those of the inflammatory cells we established an in vitro E. faecalis infection model system using the gastric carcinoma cell line MKN74. Total ROS and superoxide was measured by fluorescence microscopy. Cellular oxygen consumption was characterized non-invasively using XF24 microplate based respirometry. Gene expression was examined by microarray, and response pathways were identified by Gene Set Analysis (GSA). Selected gene transcripts were verified by quantitative real-time polymerase chain reaction (qRT-PCR). Mitochondrial mutations were determined by sequencing. Infection of MKN74 cells with E. faecalis induced intracellular ROS production through a pathway independent of oxidative phosphorylation (oxphos). Furthermore, E. faecalis infection induced mitochondrial DNA instability. Following infection, genes coding for inflammatory response proteins were transcriptionally up-regulated while DNA damage repair and cell cycle control genes were down-regulated. Cell growth slowed down when infected with viable E. faecalis and responded in a dose dependent manner to E. faecalis lysate. Infection by E. faecalis induced an oxphos-independent intracellular ROS response and damaged the mitochondrial genome in gastric cell culture. Finally the bacteria induced an NF-κB inflammatory response as well as impaired DNA damage response and cell cycle control gene expression. Array Express accession number E-MEXP-3496.

In-Vivo Real-Time Control of Protein Expression from Endogenous and Synthetic Gene Networks

PubMed Central

Orabona, Emanuele; De Stefano, Luca; Ferry, Mike; Hasty, Jeff; di Bernardo, Mario; di Bernardo, Diego

2014-01-01

We describe an innovative experimental and computational approach to control the expression of a protein in a population of yeast cells. We designed a simple control algorithm to automatically regulate the administration of inducer molecules to the cells by comparing the actual protein expression level in the cell population with the desired expression level. We then built an automated platform based on a microfluidic device, a time-lapse microscopy apparatus, and a set of motorized syringes, all controlled by a computer. We tested the platform to force yeast cells to express a desired fixed, or time-varying, amount of a reporter protein over thousands of minutes. The computer automatically switched the type of sugar administered to the cells, its concentration and its duration, according to the control algorithm. Our approach can be used to control expression of any protein, fused to a fluorescent reporter, provided that an external molecule known to (indirectly) affect its promoter activity is available. PMID:24831205

Constructing of DNA vectors with controlled nanosize and single dispersion by block copolymer coating gold nanoparticles as template assembly

NASA Astrophysics Data System (ADS)

Li, Junbo; Wu, Wenlan; Gao, Jiayu; Liang, Ju; Zhou, Huiyun; Liang, Lijuan

2017-03-01

Synthesized vectors with nanoscale size and stable colloid dispersion are highly desirable for improving gene delivery efficiency. Here, a core-shell template particle was constructed with polyethylene glycol- b-poly1-(3-aminopropyl)-3-(2-methacryloyloxy propylimidazolium bromine) (PEG- b-PAMPImB) coating gold nanoparticles (PEG- b-PAMPImB-@-Au NPs) for loading DNA and delivering in vitro. Data from transmission electron microscopy (TEM) and dynamic light scattering (DLS) suggest that these nanoplexes, by forming an electrostatic complex with DNA at the inner PAMPImB shell, offer steric protection for the outer PEG corona leading to single dispersion and small size. Notably, higher colloid stability and lower cytotoxicity were achieved with these nanoplexes when compared with PAMPImB monolayer-coated gold nanoparticles (Au NPs). Confocal laser scanning microscopy and intracellular trafficking TEM further indicate that the nanoplexes can translocate across the cell membrane and partly enter the nucleus for high efficient expression. Thus, template assembly represents a promising approach to control the size and colloid stability of gene vectors and ensure safety and efficiency of DNA delivery.

In Vitro and In Vivo Activities of Pterostilbene against Candida albicans Biofilms

PubMed Central

Li, De-Dong; Zhao, Lan-Xue; Mylonakis, Eleftherios; Hu, Gan-Hai; Zou, Yong; Huang, Tong-Kun; Yan, Lan

2014-01-01

Pterostilbene (PTE) is a stilbene-derived phytoalexin that originates from several natural plant sources. In this study, we evaluated the activity of PTE against Candida albicans biofilms and explored the underlying mechanisms. In 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assays, biofilm biomass measurement, confocal laser scanning microscopy, and scanning electron microscopy, we found that ≤16 μg/ml PTE had a significant effect against C. albicans biofilms in vitro, while it had no fungicidal effect on planktonic C. albicans cells, which suggested a unique antibiofilm effect of PTE. Then we found that PTE could inhibit biofilm formation and destroy the maintenance of mature biofilms. At 4 μg/ml, PTE decreased cellular surface hydrophobicity (CSH) and suppressed hyphal formation. Gene expression microarrays and real-time reverse transcription-PCR showed that exposure of C. albicans to 16 μg/ml PTE altered the expression of genes that function in morphological transition, ergosterol biosynthesis, oxidoreductase activity, and cell surface and protein unfolding processes (heat shock proteins). Filamentation-related genes, especially those regulated by the Ras/cyclic AMP (cAMP) pathway, including ECE1, ALS3, HWP1, HGC1, and RAS1 itself, were downregulated upon PTE treatment, indicating that the antibiofilm effect of PTE was related to the Ras/cAMP pathway. Then, we found that the addition of exogenous cAMP reverted the PTE-induced filamentous growth defect. Finally, with a rat central venous catheter infection model, we confirmed the in vivo activity of PTE against C. albicans biofilms. Collectively, PTE had strong activities against C. albicans biofilms both in vitro and in vivo, and these activities were associated with the Ras/cAMP pathway. PMID:24514088

G-fibre cell wall development in willow stems during tension wood induction

PubMed Central

Gritsch, Cristina; Wan, Yongfang; Mitchell, Rowan A. C.; Shewry, Peter R.; Hanley, Steven J.; Karp, Angela

2015-01-01

Willows (Salix spp.) are important as a potential feedstock for bioenergy and biofuels. Previous work suggested that reaction wood (RW) formation could be a desirable trait for biofuel production in willows as it is associated with increased glucose yields, but willow RW has not been characterized for cell wall components. Fasciclin-like arabinogalactan (FLA) proteins are highly up-regulated in RW of poplars and are considered to be involved in cell adhesion and cellulose biosynthesis. COBRA genes are involved in anisotropic cell expansion by modulating the orientation of cellulose microfibril deposition. This study determined the temporal and spatial deposition of non-cellulosic polysaccharides in cell walls of the tension wood (TW) component of willow RW and compared it with opposite wood (OW) and normal wood (NW) using specific antibodies and confocal laser scanning microscopy and transmission electron microscopy. In addition, the expression patterns of an FLA gene (SxFLA12) and a COBRA-like gene (SxCOBL4) were compared using RNA in situ hybridization. Deposition of the non-cellulosic polysaccharides (1–4)-β-D-galactan, mannan and de-esterified homogalacturonan was found to be highly associated with TW, often with the G-layer itself. Of particular interest was that the G-layer itself can be highly enriched in (1–4)-β-D-galactan, especially in G-fibres where the G-layer is still thickening, which contrasts with previous studies in poplar. Only xylan showed a similar distribution in TW, OW, and NW, being restricted to the secondary cell wall layers. SxFLA12 and SxCOBL4 transcripts were specifically expressed in developing TW, confirming their importance. A model of polysaccharides distribution in developing willow G-fibre cells is presented. PMID:26220085

Covalently bound DNA on naked iron oxide nanoparticles: Intelligent colloidal nano-vector for cell transfection.

PubMed

Magro, Massimiliano; Martinello, Tiziana; Bonaiuto, Emanuela; Gomiero, Chiara; Baratella, Davide; Zoppellaro, Giorgio; Cozza, Giorgio; Patruno, Marco; Zboril, Radek; Vianello, Fabio

2017-11-01

Conversely to common coated iron oxide nanoparticles, novel naked surface active maghemite nanoparticles (SAMNs) can covalently bind DNA. Plasmid (pDNA) harboring the coding gene for GFP was directly chemisorbed onto SAMNs, leading to a novel DNA nanovector (SAMN@pDNA). The spontaneous internalization of SAMN@pDNA into cells was compared with an extensively studied fluorescent SAMN derivative (SAMN@RITC). Moreover, the transfection efficiency of SAMN@pDNA was evaluated and explained by computational model. SAMN@pDNA was prepared and characterized by spectroscopic and computational methods, and molecular dynamic simulation. The size and hydrodynamic properties of SAMN@pDNA and SAMN@RITC were studied by electron transmission microscopy, light scattering and zeta-potential. The two nanomaterials were tested by confocal scanning microscopy on equine peripheral blood-derived mesenchymal stem cells (ePB-MSCs) and GFP expression by SAMN@pDNA was determined. Nanomaterials characterized by similar hydrodynamic properties were successfully internalized and stored into mesenchymal stem cells. Transfection by SAMN@pDNA occurred and GFP expression was higher than lipofectamine procedure, even in the absence of an external magnetic field. A computational model clarified that transfection efficiency can be ascribed to DNA availability inside cells. Direct covalent binding of DNA on naked magnetic nanoparticles led to an extremely robust gene delivery tool. Hydrodynamic and chemical-physical properties of SAMN@pDNA were responsible of the successful uptake by cells and of the efficiency of GFP gene transfection. SAMNs are characterized by colloidal stability, excellent cell uptake, persistence in the host cells, low toxicity and are proposed as novel intelligent DNA nanovectors for efficient cell transfection. Copyright © 2017 Elsevier B.V. All rights reserved.

Immunolocalization of a Histidine-Rich Epidermal Differentiation Protein in the Chicken Supports the Hypothesis of an Evolutionary Developmental Link between the Embryonic Subperiderm and Feather Barbs and Barbules

PubMed Central

Alibardi, Lorenzo; Holthaus, Karin Brigit; Sukseree, Supawadee; Hermann, Marcela; Tschachler, Erwin

2016-01-01

The morphogenesis of feathers is a complex process that depends on a tight spatiotemporal regulation of gene expression and assembly of the protein components of mature feathers. Recent comparative genomics and gene transcription studies have indicated that genes within the epidermal differentiation complex (EDC) encode numerous structural proteins of cornifying skin cells in amniotes including birds. Here, we determined the localization of one of these proteins, termed EDMTFH (Epidermal Differentiation Protein starting with a MTF motif and rich in Histidine), which belongs to a group of EDC-encoded proteins rich in aromatic amino acid residues. We raised an antibody against an EDMTFH-specific epitope and performed immunohistochemical investigations by light microscopy and immunogold labeling by electron microscopy of chicken embryos at days 14–18 of development. EDMTFH was specifically present in the subperiderm, a transient layer of the embryonic epidermis, and in barbs and barbules of feathers. In the latter, it partially localized to bundles of so-called feather beta-keratins (corneous beta-proteins, CBPs). Cells of the embryonic periderm, the epidermis proper, and the feather sheath were immunonegative for EDMTFH. The results of this study indicate that EDMTFH may contribute to the unique mechanical properties of feathers and define EDMTFH as a common marker of the subperiderm and the feather barbules. This expression pattern of EDMTFH resembles that of epidermal differentiation cysteine-rich protein (EDCRP) and feather CBPs and is in accordance with the hypothesis that a major part of the cyclically regenerating feather follicle is topologically, developmentally and evolutionarily related to the embryonic subperiderm. PMID:27936131

PECTATE LYASE-LIKE 9 from Brassica campestris is associated with intine formation.

PubMed

Jiang, Jingjing; Yao, Lina; Yu, Youjian; Liang, Ying; Jiang, Jianxia; Ye, Nenghui; Miao, Ying; Cao, Jiashu

2014-12-01

Brassica campestris pectate lyase-like 9 (BcPLL9) was previously identified as a differentially expressed gene both in buds during late pollen developmental stage and in pistils during fertilization in Chinese cabbage. To characterize the gene's function, antisense-RNA lines of BcPLL9 (bcpll9) were constructed in Chinese cabbage. Self- and cross-fertilization experiments harvested half seed yields when bcpll9 lines were used as pollen donors. In vivo and in vitro pollen germination assays showed that nearly half of the pollen tubes in bcpll9 were irregular with shorter length and uneven surface. Aniline blue staining identified abnormal accumulation of a specific bright blue unknown material in the bcpll9 pollen portion. Scanning electron microscopy observation verified the abnormal outthrust material to be near the pollen germinal furrows. Transmission electron microscopy observation revealed the internal endintine layer was overdeveloped and predominantly occupied the intine. This abnormally formed intine likely induced the wavy structure and growth arrest of the pollen tube in half of the bcpll9 pollen grains, which resulted in less seed yields. Collectively, this study presented a novel PLL gene that has an important function in B. campestris intine formation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Pathway Analysis Hints Towards Beneficial Effects of Long-Term Vibration on Human Chondrocytes.

PubMed

Lützenberg, Ronald; Solano, Kendrick; Buken, Christoph; Sahana, Jayashree; Riwaldt, Stefan; Kopp, Sascha; Krüger, Marcus; Schulz, Herbert; Saar, Kathrin; Huebner, Norbert; Hemmersbach, Ruth; Bauer, Johann; Infanger, Manfred; Grimm, Daniela; Wehland, Markus

2018-06-27

Spaceflight negatively influences the function of cartilage tissue in vivo. In vitro human chondrocytes exhibit an altered gene expression of inflammation markers after a two-hour exposure to vibration. Little is known about the impact of long-term vibration on chondrocytes. Human cartilage cells were exposed for up to 24 h (VIB) on a specialised vibration platform (Vibraplex) simulating the vibration profile which occurs during parabolic flights and compared to static control conditions (CON). Afterwards, they were investigated by phase-contrast microscopy, rhodamine phalloidin staining, microarray analysis, qPCR and western blot analysis. Morphological investigations revealed no changes between CON and VIB chondrocytes. F-Actin staining showed no alterations of the cytoskeleton in VIB compared with CON cells. DAPI and TUNEL staining did not identify apoptotic cells. ICAM-1 was elevated and vimentin, beta-tubulin and osteopontin proteins were significantly reduced in VIB compared to CON cells. qPCR of cytoskeletal genes, ITGB1, SOX3, SOX5, SOX9 did not reveal differential regulations. Microarray analysis detected 13 differentially expressed genes, mostly indicating unspecific stimulations. Pathway analyses demonstrated interactions of PSMD4 and CNOT7 with ICAM. Long-term vibration did not damage human chondrocytes in vitro. The reduction of osteopontin protein and the down-regulation of PSMD4 and TBX15 gene expression suggest that in vitro long-term vibration might even positively influence cultured chondrocytes. © 2018 The Author(s). Published by S. Karger AG, Basel.

Expression of adhesion and extracellular matrix genes in human blastocysts upon attachment in a 2D co-culture system.

PubMed

Aberkane, A; Essahib, W; Spits, C; De Paepe, C; Sermon, K; Adriaenssens, T; Mackens, S; Tournaye, H; Brosens, J J; Van de, Velde H

2018-05-26

What are the changes in human embryos, in terms of morphology and gene expression, upon attachment to endometrial epithelial cells? Apposition and adhesion of human blastocysts to endometrial epithelial cells are predominantly initiated at the embryonic pole and these steps are associated with changes in expression of adhesion and extracellular matrix (ECM) genes in the embryo. Both human and murine embryos have been co-cultured with Ishikawa cells, although embryonic gene expression associated with attachment has not yet been investigated in an in-vitro implantation model. Vitrified human blastocysts were warmed and co-cultured for up to 48 h with Ishikawa cells, a model cell line for receptive endometrial epithelium. Six-days post fertilisation (6dpf) human embryos were co-cultured with Ishikawa cells for 12 h, 24 h (7dpf) or 48 h (8dpf) and attachment rate and morphological development investigated. Expression of 84 adhesion and ECM genes was analysed by quantitative PCR. Immunofluorescence microscopy was used to assess the expression of three informative genes at the protein level. Data are reported on 115 human embryos. Mann-Whitney U was used for statistical analysis between two groups, with P < 0.05 considered significant. The majority of embryos attached to Ishikawa cells at the level of the polar trophectoderm; 41% of co-cultured embryos were loosely attached after 12 h and 86% firmly attached after 24 h. Outgrowth of hCG-positive embryonic cells at 8dpf indicated differentiation of trophectoderm into invasive syncytiotrophoblast. Gene expression analysis was performed on loosely attached and unattached embryos co-cultured with Ishikawa cells for 12 h. In contrast to unattached embryos, loosely attached embryos expressed THBS1, TNC, COL12A1, CTNND2, ITGA3, ITGAV, and LAMA3 and had significantly higher CD44 and TIMP1 transcript levels (P = 0.014 and P = 0.029, respectively). LAMA3, THBS1 and TNC expression was validated at the protein level in firmly attached 7dpf embryos. Thrombospondin 1 (THBS1) resided in the cytoplasm of embryonic cells whereas laminin subunit alpha 3 (LAMA3) and tenascin C (TNC) were expressed on the cell surface of trophectoderm cells. Incubation with a neutralizing TNC antibody did not affect the rate of embryo attachment or hCG secretion. None. This in-vitro study made use of an endometrial adenocarcinoma cell line to mimic receptive luminal epithelium. Also, the number of embryos was limited. Contamination of recovered embryos with Ishikawa cells was unlikely based on their differential gene expression profiles. Taken together, we provide a 'proof of concept' that initiation of the implantation process coincides with the induction of specific embryonic genes. Genome-wide expression profiling of a larger sample set may provide insights into the molecular embryonic pathways underlying successful or failed implantation. A.A. was supported by a grant from the "Instituut voor Innovatie door Wetenschap en Technologie" (IWT, 121716, Flanders, Belgium). This work was supported by the "Wetenschappelijk Fonds Willy Gepts" (WFWG G142 and G170, Universitair Ziekenhuis Brussel). The authors declare no conflict of interest.

Characterization of Novel Plant Symbiosis Mutants Using a New Multiple Gene-Expression Reporter Sinorhizobium meliloti Strain

PubMed Central

Lang, Claus; Smith, Lucinda S.; Haney, Cara H.; Long, Sharon R.

2018-01-01

The formation of nitrogen fixing root nodules by Medicago truncatula and Sinorhizobium meliloti requires communication between both organisms and coordinated differentiation of plant and bacterial cells. After an initial signal exchange, the bacteria invade the tissue of the growing nodule via plant-derived tubular structures, called infection threads. The bacteria are released from the infection threads into invasion-competent plant cells, where they differentiate into nitrogen-fixing bacteroids. Both organisms undergo dramatic transcriptional, metabolic and morphological changes during nodule development. To identify plant processes that are essential for the formation of nitrogen fixing nodules after nodule development has been initiated, large scale mutageneses have been conducted to discover underlying plant symbiosis genes. Such screens yield numerous uncharacterized plant lines with nitrogen fixation deficient nodules. In this study, we report construction of a S. meliloti strain carrying four distinct reporter constructs to reveal stages of root nodule development. The strain contains a constitutively expressed lacZ reporter construct; a PexoY-mTFP fusion that is expressed in infection threads but not in differentiated bacteroids; a PbacA-mcherry construct that is expressed in infection threads and during bacteroid differentiation; and a PnifH-uidA construct that is expressed during nitrogen fixation. We used this strain together with fluorescence microscopy to study nodule development over time in wild type nodules and to characterize eight plant mutants from a fast neutron bombardment screen. Based on the signal intensity and the localization patterns of the reporter genes, we grouped mutants with similar phenotypes and placed them in a developmental context. PMID:29467773

Characterization of Novel Plant Symbiosis Mutants Using a New Multiple Gene-Expression Reporter Sinorhizobium meliloti Strain.

PubMed

Lang, Claus; Smith, Lucinda S; Long, Sharon R

2018-01-01

The formation of nitrogen fixing root nodules by Medicago truncatula and Sinorhizobium meliloti requires communication between both organisms and coordinated differentiation of plant and bacterial cells. After an initial signal exchange, the bacteria invade the tissue of the growing nodule via plant-derived tubular structures, called infection threads. The bacteria are released from the infection threads into invasion-competent plant cells, where they differentiate into nitrogen-fixing bacteroids. Both organisms undergo dramatic transcriptional, metabolic and morphological changes during nodule development. To identify plant processes that are essential for the formation of nitrogen fixing nodules after nodule development has been initiated, large scale mutageneses have been conducted to discover underlying plant symbiosis genes. Such screens yield numerous uncharacterized plant lines with nitrogen fixation deficient nodules. In this study, we report construction of a S. meliloti strain carrying four distinct reporter constructs to reveal stages of root nodule development. The strain contains a constitutively expressed lacZ reporter construct; a P exoY -mTFP fusion that is expressed in infection threads but not in differentiated bacteroids; a P bacA -mcherry construct that is expressed in infection threads and during bacteroid differentiation; and a P nifH -uidA construct that is expressed during nitrogen fixation. We used this strain together with fluorescence microscopy to study nodule development over time in wild type nodules and to characterize eight plant mutants from a fast neutron bombardment screen. Based on the signal intensity and the localization patterns of the reporter genes, we grouped mutants with similar phenotypes and placed them in a developmental context.

A novel recombinant pseudorabies virus expressing parvovirus VP2 gene: Immunogenicity and protective efficacy in swine

PubMed Central

2011-01-01

Background Porcine parvovirus (PPV) VP2 gene has been successfully expressed in many expression systems resulting in self-assembly of virus-like particles (VLPs) with similar morphology to the native capsid. Here, a pseudorabies virus (PRV) system was adopted to express the PPV VP2 gene. Methods A recombinant PRV SA215/VP2 was obtained by homologous recombination between the vector PRV viral DNA and a transfer plasmid. Then recombinant virus was purified with plaque purification, and its identity confirmed by PCR amplification, Western blot and indirect immunofluorescence (IFA) analyses. Electronic microscopy of PRV SA215/VP2 confirmed self-assembly of both pseudorabies virus and VLPs from VP2 protein. Results Immunization of piglets with recombinant virus elicited PRV-specific and PPV-specific humoral immune responses and provided complete protection against a lethal dose of PRV challenges. Gilts immunized with recombinant viruses induced PPV-specific antibodies, and significantly reduced the mortality rate of (1 of 28) following virulent PPV challenge compared with the control (7 of 31). Furthermore, PPV virus DNA was not detected in the fetuses of recombinant virus immunized gilts. Conclusions In this study, a recombinant PRV SA215/VP2 virus expressing PPV VP2 protein was constructed using PRV SA215 vector. The safety, immunogenicity, and protective efficacy of the recombinant virus were demonstrated in piglets and primiparous gilts. This recombinant PRV SA215/VP2 represents a suitable candidate for the development of a bivalent vaccine against both PRV and PPV infection. PMID:21679423

A standardized extract of Butea monosperma (Lam.) flowers suppresses the IL-1β-induced expression of IL-6 and matrix-metalloproteases by activating autophagy in human osteoarthritis chondrocytes.

PubMed

Ansari, Mohammad Y; Khan, Nazir M; Haqqi, Tariq M

2017-12-01

Osteoarthritis (OA) is a leading cause of joint dysfunction, disability and poor quality of life in the affected population. The underlying mechanism of joint dysfunction involves increased oxidative stress, inflammation, high levels of cartilage extracellular matrix degrading proteases and decline in autophagy-a mechanism of cellular defense. There is no disease modifying therapies currently available for OA. Different parts of the Butea monosperma (Lam.) plant have widely been used in the traditional Indian Ayurvedic medicine system for the treatment of various human diseases including inflammatory conditions. Here we studied the chondroprotective effect of hydromethanolic extract of Butea monosperma (Lam.) flowers (BME) standardized to the concentration of Butein on human OA chondrocytes stimulated with IL-1β. The hydromethanolic extract of Butea monosperma (Lam.) (BME) was prepared with 70% methanol-water mixer using Soxhlet. Chondrocytes viability after BME treatment was measured by MTT assay. Gene expression levels were determined by quantitative polymerase chain reaction (qPCR) using TaqMan assays and immunoblotting with specific antibodies. Autophagy activation was determined by measuring the levels of microtubule associated protein 1 light chain 3-II (LC3-II) by immunoblotting and visualization of autophagosomes by transmission electron and confocal microscopy. BME was non-toxic to the OA chondrocytes at the doses employed and suppressed the IL-1β induced expression of inerleukin-6 (IL-6) and matrix metalloprotease-3 (MMP-3), MMP-9 and MMP-13. BME enhanced autophagy in chondrocytes as determined by measuring the levels of LC3-II by immunoblotting and increased number of autophagosomes in BME treated chondrocytes by transmission electron microscopy and confocal microscopy. BME upregulated the expression of several autophagy related genes and increased the autophagy flux in human OA chondrocytes under pathological conditions. Further analysis revealed that BME activated autophagy in chondrocytes via inhibition of mammalian target of rapamycin (mTOR) pathway. Of importance is our finding that BME-mediated suppression of IL-1β induced expression of IL-6, MMP-3, -9, and -13 was autophagy dependent and was abrogated by inhibition of autophagy. The above results show that the Butea monosperma (Lam.) extract has strong potential to activate autophagy and suppress IL-1β induced expression of IL-6 and MMP-3, -9 and -13 in human OA chondrocytes. This study shows that BME or compounds derived from BME can be developed as safe and effective chondroprotective agent(s) that function by activating autophagy to suppress the expression of inflammatory and catabolic factors associated with OA pathogenesis. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Prone to fix: Resilience of the active nitrogen-fixing rice root microbiome

NASA Astrophysics Data System (ADS)

Hurek, Thomas; Sabale, Mugdha; Sarkar, Abhijit; Pees, Tobias; Reinhold-Hurek, Barbara

2016-04-01

Due to water consumption, many lowland rice areas in Asia are undergoing a transition that involves adoption of new management strategies, with crop rotations encompassing a non-flooded crop, including maize. Shifting from flooded to non-flooded cropping is likely to affect microbial nitrogen cycling. For analysis of the root-associated microbiome of rice and maize in response to flooding or nitrogen fertilizer, we combine methods of microbial ecology (Next-Generation sequencing of amplicons), and a reductionist approach with pure cultures of the endophytic diazotroph Azoarus sp.. Field plots of the ICON project (Introducing non-flooded crops in rice-dominated landscapes: Impact on Carbon, nitrogen and water budgets) at the International Rice Research Institute in the Philippines were analyzed. Root-associated activity of nitrogenase gene expression was assessed by quantitative RT-PCR of nifH. For rice, expression levels were surprisingly stable, in response to non-flooded versus flooded conditions, or in response to conventional nitrogen fertilizer applications versus lack of N-fertilizer. In contrast, the active diazotrophic population of maize roots was not resistant to N-fertilization, nifH expression strongly decreased. Concordant changes in the diazotrophic resident or active communities were detected by nifH amplicon sequence analysis, based on bacterial DNA or mRNA, respectively. For high-resolution analyses of the endobiome in gnotobiotic culture, we developed a dual fluorescence reporter system for Azoarcus sp. BH72 which allows to quantify and visualize epi- and endophytic gene expression by concfocal microscopy (CLSM). This allowed us to demonstrate sites of active nitrogen fixation (gene expression) in association with rice roots. We confirmed that at low nitrogen fertilizer levels, endophytic nifH gene expression persisted in rice roots, while it was repressed in maize roots. This supports our observation of remarkable stability of nitrogen fixation in association with rice roots.

Analysis of Subcellular Prefoldin 1 Redistribution During Rabies Virus Infection

PubMed Central

Zhang, Jinyang; Han, Qinqin; Song, Yuzhu; Chen, Qiang; Xia, Xueshan

2015-01-01

Background: Rabies virus (RABV) is one of the old deadly zoonotic viruses. It attacks the central nervous system and causes acute encephalitis in humans and animals. Host factors are known to be essential for virus infection and replication in cells. The identification of the key host factors required for RABV infection may provide important information on RABV replication and may provide new potential targets for RABV drug discovery. Objectives: This study aimed to investigate the change in the subcellular distribution and expression of the host protein Prefoldin subunit 1 (PFDN1) in RABV-infected cells and the viral expression of plasmids in the transfected cells. Materials and Methods: Mouse Neuro-2a (N2a) cells were infected by RABV or transfected with the plasmids of the nucleoprotein (N) and/or phosphoprotein (P) gene of RABV. The subcellular distribution of PFDN1 was analyzed by confocal microscopy, and the transcription levels of PFDN1 in the N and/or P gene of the RABV-transfected or RABV-infected N2a cells were assessed via real-time quantitative polymerase chain reaction. Results: Confocal microscopy showed that PFDN1 was colocalized with the N protein of RABV in the infected N2a cells and was mainly recruited to the characteristic Negri-Body-Like (NBL) structures in the cytoplasm, as well as the cotransfection of the N and P genes of RABV. The transcription of PFDN1 in the RABV-infected N2a cells was upregulated, whereas the transfection of the N and/or P genes did not result in the upregulation of PFDN1. Conclusions: The results of this work demonstrated that the subcellular distribution of PFDN1 was altered in the RABV-infected N2a cells and colocalized with the N protein of RABV in the NBL structures. PMID:26421138

A Circadian Clock Gene, Cry, Affects Heart Morphogenesis and Function in Drosophila as Revealed by Optical Coherence Microscopy

PubMed Central

Zeng, Xianxu; Tate, Rebecca E.; McKee, Mary L.; Capen, Diane E.; Zhang, Zhan; Tanzi, Rudolph E.; Zhou, Chao

2015-01-01

Circadian rhythms are endogenous, entrainable oscillations of physical, mental and behavioural processes in response to local environmental cues such as daylight, which are present in the living beings, including humans. Circadian rhythms have been related to cardiovascular function and pathology. However, the role that circadian clock genes play in heart development and function in a whole animal in vivo are poorly understood. The Drosophila cryptochrome (dCry) is a circadian clock gene that encodes a major component of the circadian clock negative feedback loop. Compared to the embryonic stage, the relative expression levels of dCry showed a significant increase (>100-fold) in Drosophila during the pupa and adult stages. In this study, we utilized an ultrahigh resolution optical coherence microscopy (OCM) system to perform non-invasive and longitudinal analysis of functional and morphological changes in the Drosophila heart throughout its post-embryonic lifecycle for the first time. The Drosophila heart exhibited major morphological and functional alterations during its development. Notably, heart rate (HR) and cardiac activity period (CAP) of Drosophila showed significant variations during the pupa stage, when heart remodeling took place. From the M-mode (2D + time) OCM images, cardiac structural and functional parameters of Drosophila at different developmental stages were quantitatively determined. In order to study the functional role of dCry on Drosophila heart development, we silenced dCry by RNAi in the Drosophila heart and mesoderm, and quantitatively measured heart morphology and function in those flies throughout its development. Silencing of dCry resulted in slower HR, reduced CAP, smaller heart chamber size, pupal lethality and disrupted posterior segmentation that was related to increased expression of a posterior compartment protein, wingless. Collectively, our studies provided novel evidence that the circadian clock gene, dCry, plays an essential role in heart morphogenesis and function. PMID:26348211

CCDC65 Mutation Causes Primary Ciliary Dyskinesia with Normal Ultrastructure and Hyperkinetic Cilia

PubMed Central

Horani, Amjad; Brody, Steven L.; Ferkol, Thomas W.; Shoseyov, David; Wasserman, Mollie G.; Ta-shma, Asaf; Wilson, Kate S.; Bayly, Philip V.; Amirav, Israel; Cohen-Cymberknoh, Malena; Dutcher, Susan K.; Elpeleg, Orly; Kerem, Eitan

2013-01-01

Background Primary ciliary dyskinesia (PCD) is a genetic disorder characterized by impaired ciliary function, leading to chronic sinopulmonary disease. The genetic causes of PCD are still evolving, while the diagnosis is often dependent on finding a ciliary ultrastructural abnormality and immotile cilia. Here we report a novel gene associated with PCD but without ciliary ultrastructural abnormalities evident by transmission electron microscopy, but with dyskinetic cilia beating. Methods Genetic linkage analysis was performed in a family with a PCD subject. Gene expression was studied in Chlamydomonas reinhardtii and human airway epithelial cells, using RNA assays and immunostaining. The phenotypic effects of candidate gene mutations were determined in primary culture human tracheobronchial epithelial cells transduced with gene targeted shRNA sequences. Video-microscopy was used to evaluate cilia motion. Results A single novel mutation in CCDC65, which created a termination codon at position 293, was identified in a subject with typical clinical features of PCD. CCDC65, an orthologue of the Chlamydomonas nexin-dynein regulatory complex protein DRC2, was localized to the cilia of normal nasal epithelial cells but was absent in those from the proband. CCDC65 expression was up-regulated during ciliogenesis in cultured airway epithelial cells, as was DRC2 in C. reinhardtii following deflagellation. Nasal epithelial cells from the affected individual and CCDC65-specific shRNA transduced normal airway epithelial cells had stiff and dyskinetic cilia beating patterns compared to control cells. Moreover, Gas8, a nexin-dynein regulatory complex component previously identified to associate with CCDC65, was absent in airway cells from the PCD subject and CCDC65-silenced cells. Conclusion Mutation in CCDC65, a nexin-dynein regulatory complex member, resulted in a frameshift mutation and PCD. The affected individual had altered cilia beating patterns, and no detectable ultrastructural defects of the ciliary axoneme, emphasizing the role of the nexin-dynein regulatory complex and the limitations of certain methods for PCD diagnosis. PMID:23991085

Assessment of genetically engineered Trabulsiella odontotermitis as a 'Trojan Horse' for paratransgenesis in termites.

PubMed

Tikhe, Chinmay Vijay; Martin, Thomas M; Howells, Andréa; Delatte, Jennifer; Husseneder, Claudia

2016-09-05

The Formosan subterranean termite, Coptotermes formosanus is an invasive urban pest in the Southeastern USA. Paratransgenesis using a microbe expressed lytic peptide that targets the termite gut protozoa is currently being developed for the control of Formosan subterranean termites. In this study, we evaluated Trabulsiella odontotermitis, a termite-specific bacterium, for its potential to serve as a 'Trojan Horse' for expression of gene products in termite colonies. We engineered two strains of T. odontotermitis, one transformed with a constitutively expressed GFP plasmid and the other engineered at the chromosome with a Kanamycin resistant gene using a non- disruptive Tn7 transposon. Both strains were fed to termites from three different colonies. Fluorescent microscopy confirmed that T. odontotermitis expressed GFP in the gut and formed a biofilm in the termite hindgut. However, GFP producing bacteria could not be isolated from the termite gut after 2 weeks. The feeding experiment with the chromosomally engineered strain demonstrated that T. odontotermitis was maintained in the termite gut for at least 21 days, irrespective of the termite colony. The bacteria persisted in two termite colonies for at least 36 days post feeding. The experiment also confirmed the horizontal transfer of T. odontotermitis amongst nest mates. Overall, we conclude that T. odontotermitis can serve as a 'Trojan Horse' for spreading gene products in termite colonies. This study provided proof of concept and laid the foundation for the future development of genetically engineered termite gut bacteria for paratransgenesis based termite control.

The Glyoxylate Cycle in an Arbuscular Mycorrhizal Fungus. Carbon Flux and Gene Expression

PubMed Central

Lammers, Peter J.; Jun, Jeongwon; Abubaker, Jehad; Arreola, Raul; Gopalan, Anjali; Bago, Berta; Hernandez-Sebastia, Cinta; Allen, James W.; Douds, David D.; Pfeffer, Philip E.; Shachar-Hill, Yair

2001-01-01

The arbuscular mycorrhizal (AM) symbiosis is responsible for huge fluxes of photosynthetically fixed carbon from plants to the soil. Lipid, which is the dominant form of stored carbon in the fungal partner and which fuels spore germination, is made by the fungus within the root and is exported to the extraradical mycelium. We tested the hypothesis that the glyoxylate cycle is central to the flow of carbon in the AM symbiosis. The results of 13C labeling of germinating spores and extraradical mycelium with 13C2-acetate and 13C2-glycerol and analysis by nuclear magnetic resonance spectroscopy indicate that there are very substantial fluxes through the glyoxylate cycle in the fungal partner. Full-length sequences obtained by polymerase chain reaction from a cDNA library from germinating spores of the AM fungus Glomus intraradices showed strong homology to gene sequences for isocitrate lyase and malate synthase from plants and other fungal species. Quantitative real-time polymerase chain reaction measurements show that these genes are expressed at significant levels during the symbiosis. Glyoxysome-like bodies were observed by electron microscopy in fungal structures where the glyoxylate cycle is expected to be active, which is consistent with the presence in both enzyme sequences of motifs associated with glyoxysomal targeting. We also identified among several hundred expressed sequence tags several enzymes of primary metabolism whose expression during spore germination is consistent with previous labeling studies and with fluxes into and out of the glyoxylate cycle. PMID:11706207

Farnesol-induced apoptosis in Candida albicans.

PubMed

Shirtliff, Mark E; Krom, Bastiaan P; Meijering, Roelien A M; Peters, Brian M; Zhu, Jingsong; Scheper, Mark A; Harris, Megan L; Jabra-Rizk, Mary Ann

2009-06-01

Farnesol, a precursor in the isoprenoid/sterol pathway, was recently identified as a quorum-sensing molecule produced by the fungal pathogen Candida albicans. Farnesol is involved in the inhibition of germination and biofilm formation by C. albicans and can be cytotoxic at certain concentrations. In addition, we have shown that farnesol can trigger apoptosis in mammalian cells via the classical apoptotic pathways. In order to elucidate the mechanism behind farnesol cytotoxicity in C. albicans, the response to farnesol was investigated, using proteomic analysis. Global protein expression profiles demonstrated significant changes in protein expression resulting from farnesol exposure. Among the downregulated proteins were those involved in metabolism, glycolysis, protein synthesis, and mitochondrial electron transport and the respiratory chain, whereas proteins involved in folding, protection against environmental and oxidative stress, actin cytoskeleton reorganization, and apoptosis were upregulated. Cellular changes that accompany apoptosis (regulated cell death) were further analyzed using fluorescent microscopy and gene expression analysis. The results indicated reactive oxygen species accumulation, mitochondrial degradation, and positive terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) in the farnesol-exposed cells concurrent with increased expression of antioxidant-encoding and drug response genes. More importantly, the results demonstrated farnesol-induced upregulation of the caspase gene MCA1 and the intracellular presence of activated caspases. In conclusion, this study demonstrated that farnesol promotes apoptosis in C. albicans through caspase activation, implying an important physiological role for farnesol in the fungal cell life cycle with important implications for adaptation and survival.

Cell junction protein armadillo repeat gene deleted in velo-cardio-facial syndrome is expressed in the skin and colocalizes with autoantibodies of patients affected by a new variant of endemic pemphigus foliaceus in Colombia.

PubMed

Abreu-Velez, Ana Maria; Yi, Hong; Howard, Michael S

2017-10-01

We previously described a new variant of endemic pemphigus foliaceus in El Bagre, Colombia, South America (El Bagre-EPF, or pemphigus Abreu-Manu). El Bagre-EPF differs from other types of EPF clinically, epidemiologically, immunologically and in its target antigens. We reported the presence of patient autoantibodies colocalizing with armadillo repeat gene deleted in velo-cardio-facial syndrome (ARVCF), a catenin cell junction protein colocalizing with El Bagre-EPF autoantibodies in the heart and within pilosebaceous units along their neurovascular supply routes. Here we investigate the presence of ARVCF in skin and its possibility as a cutaneous El Bagre-EPF antigen. We used a case-control study, testing sera of 45 patients and 45 controls via direct and indirect immunofluorescence (DIF/IIF), confocal microscopy, immunoelectron microscopy and immunoblotting for the presence of ARVCF and its relationship with El Bagre-EPF autoantibodies in the skin. We also immunoadsorbed samples with desmoglein 1 (Dsg1) ectodomain (El Bagre-EPF antigen) by incubating with the positive ARVCF samples from DIF and IIF. ARVCF was expressed in all the samples from the cases and controls. Immunoadsorption with Dsg1 on positive ARVCF immunofluorescence DIF/IIF cases showed that the immune response was present against non-desmoglein 1 antigen(s). Overall, 40/45 patients showed colocalization of their autoantibodies with ARVCF in the epidermis; no controls from the endemic area displayed colocalization. We demonstrate that ARVCF is expressed in many areas of human skin, and colocalizes with the majority of El Bagre-EPF autoantibodies as a putative antigen.

Cell junction protein armadillo repeat gene deleted in velo-cardio-facial syndrome is expressed in the skin and colocalizes with autoantibodies of patients affected by a new variant of endemic pemphigus foliaceus in Colombia

PubMed Central

Abreu-Velez, Ana Maria; Yi, Hong; Howard, Michael S.

2017-01-01

Background We previously described a new variant of endemic pemphigus foliaceus in El Bagre, Colombia, South America (El Bagre-EPF, or pemphigus Abreu-Manu). El Bagre-EPF differs from other types of EPF clinically, epidemiologically, immunologically and in its target antigens. We reported the presence of patient autoantibodies colocalizing with armadillo repeat gene deleted in velo-cardio-facial syndrome (ARVCF), a catenin cell junction protein colocalizing with El Bagre-EPF autoantibodies in the heart and within pilosebaceous units along their neurovascular supply routes. Here we investigate the presence of ARVCF in skin and its possibility as a cutaneous El Bagre-EPF antigen. Methods We used a case-control study, testing sera of 45 patients and 45 controls via direct and indirect immunofluorescence (DIF/IIF), confocal microscopy, immunoelectron microscopy and immunoblotting for the presence of ARVCF and its relationship with El Bagre-EPF autoantibodies in the skin. We also immunoadsorbed samples with desmoglein 1 (Dsg1) ectodomain (El Bagre-EPF antigen) by incubating with the positive ARVCF samples from DIF and IIF. Results ARVCF was expressed in all the samples from the cases and controls. Immunoadsorption with Dsg1 on positive ARVCF immunofluorescence DIF/IIF cases showed that the immune response was present against non-desmoglein 1 antigen(s). Overall, 40/45 patients showed colocalization of their autoantibodies with ARVCF in the epidermis; no controls from the endemic area displayed colocalization. Conclusions We demonstrate that ARVCF is expressed in many areas of human skin, and colocalizes with the majority of El Bagre-EPF autoantibodies as a putative antigen. PMID:29214101

miR-99 inhibits cervical carcinoma cell proliferation by targeting TRIB2

PubMed Central

XIN, JIA-XUAN; YUE, ZHEN; ZHANG, SHUAI; JIANG, ZHONG-HUA; WANG, PING-YU; LI, YOU-JIE; PANG, MIN; XIE, SHU-YANG

2013-01-01

MicroRNAs (miRNAs) have significant roles in cell processes, including proliferation, apoptosis and stress responses. To investigate the involvement of miR-99 in the inhibition of HeLa cell proliferation, an miR-99 gene expression vector (pU6.1/miR-99), which overexpressed miR-99 in HeLa cells after transient transfection, was constructed. The expression of miR-99 was detected by qPCR. Cell proliferation and apoptosis were analyzed by cell viability, proliferation and apoptosis assays, as well as by electron microscopy. The results showed that overexpression of miR-99 in HeLa cells increased the HeLa cell mortality rate. Moreover, miR-99 overexpression was able to markedly inhibit HeLa cell proliferation according to the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The cell apoptosis rate was significantly higher in pU6.1/miR-99-treated cells compared with that in the control cultures. Increases in intracellular electron density, as well as the proportion of nuclear plasma, blebbing phenomena and apoptotic bodies were observed in pU6.1/miR-99-treated cells compared with control cultures according to electron microscopy analysis. The Tribbles 2 (TRIB2) 3′-untranslated region was also observed to be targeted by miR-99 and the results further demonstrated that miR-99 was able to negatively regulate TRIB2 expression in HeLa cells The results indicate that miR-99 acts as a tumor suppressor gene in HeLa cells, establishing a theoretical basis for its application in cancer therapeutics. PMID:24137458

Nop2 is expressed during proliferation of neural stem cells and in adult mouse and human brain.

PubMed

Kosi, Nina; Alić, Ivan; Kolačević, Matea; Vrsaljko, Nina; Jovanov Milošević, Nataša; Sobol, Margarita; Philimonenko, Anatoly; Hozák, Pavel; Gajović, Srećko; Pochet, Roland; Mitrečić, Dinko

2015-02-09

The nucleolar protein 2 gene encodes a protein specific for the nucleolus. It is assumed that it plays a role in the synthesis of ribosomes and regulation of the cell cycle. Due to its link to cell proliferation, higher expression of Nop2 indicates a worse tumor prognosis. In this work we used Nop2(gt1gaj) gene trap mouse strain. While lethality of homozygous animals suggested a vital role of this gene, heterozygous animals allowed the detection of expression of Nop2 in various tissues, including mouse brain. Histochemistry, immunohistochemistry and immunoelectron microscopy techniques, applied to a mature mouse brain, human brain and on mouse neural stem cells revealed expression of Nop2 in differentiating cells, including astrocytes, as well as in mature neurons. Nop2 was detected in various regions of mouse and human brain, mostly in large pyramidal neurons. In the human, Nop2 was strongly expressed in supragranular and infragranular layers of the somatosensory cortex and in layer III of the cingulate cortex. Also, Nop2 was detected in CA1 and the subiculum of the hippocampus. Subcellular analyses revealed predominant location of Nop2 within the dense fibrillar component of the nucleolus. To test if Nop2 expression correlates to cell proliferation occurring during tissue regeneration, we induced strokes in mice by middle cerebral artery occlusion. Two weeks after stroke, the number of Nop2/nestin double positive cells in the region affected by ischemia and the periventricular zone substantially increased. Our findings suggest a newly discovered role of Nop2 in both mature neurons and in cells possibly involved in the regeneration of nervous tissue. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Participation of GATA-3 in regulation of bone healing through transcriptional upregulation of bcl-xL expression

PubMed Central

Liao, Mei-Hsiu; Lin, Pei-I; Ho, Wei-Pin; Chan, Wing P; Chen, Ta-Liang; Chen, Ruei-Ming

2017-01-01

We have previously demonstrated the expression of GATA-DNA-binding protein (GATA)-3, a transcription factor, in osteoblasts and have verified its function in transducing cell survival signaling. This translational study was further designed to evaluate the roles of GATA-3 in regulating bone healing and to explore its possible mechanisms. A metaphyseal bone defect was created in the left femurs of male ICR mice. Analysis by micro-computed topography showed that the bone volume, trabecular bone number and trabecular thickness were augmented and that the trabecular pattern factor decreased. Interestingly, immunohistological analyses showed specific expression of GATA-3 in the defect area. In addition, colocalized expression of GATA-3 and alkaline phosphatase was observed at the wound site. As the fracture healed, the amounts of phosphorylated and non-phosphorylated GATA-3 concurrently increased. Separately, GATA-3 mRNA was induced during bone healing, and, levels of Runx2 mRNA and protein were also increased. The results of confocal microscopy and co-immunoprecipitation showed an association between nuclear GATA-3 and Runx2 in the area of insult. In parallel with fracture healing, Bcl-XL mRNA was significantly triggered. A bioinformatic search revealed the existence of a GATA-3-specific DNA-binding element in the promoter region of the bcl-xL gene. Analysis by chromatin immunoprecipitation assays further demonstrated transactivation activity by which GATA-3 regulated bcl-xL gene expression. Therefore, this study shows that GATA-3 participates in the healing of bone fractures via regulating bcl-xL gene expression, owing to its association with Runx2. In the clinic, GATA-3 may be used as a biomarker for diagnoses/prognoses or as a therapeutic target for bone diseases, such as bone fractures. PMID:29170477

Vaccination with Newcastle disease virus vectored vaccine protects chickens against highly pathogenic H7 avian influenza virus.

PubMed

Schröer, Diana; Veits, Jutta; Grund, Christian; Dauber, Malte; Keil, Günther; Granzow, Harald; Mettenleiter, Thomas C; Römer-Oberdörfer, Angela

2009-06-01

A recombinant Newcastle disease virus (NDV) was engineered to express the hemagglutinin (HA) gene of avian influenza virus (AIV) subtype H7. The HA gene was inserted between the genes encoding NDV fusion and hemagglutinin-neuraminidase proteins. Within the H7 open reading frame, an NDV gene end-like sequence was eliminated by silent mutation. The expression of H7 protein was detected by western blot analysis and indirect immunofluorescence. The existence of H7 protein in the envelope of recombinant Newcastle disease virions was shown by immunoelectron microscopy. The protective efficacy of recombinant NDVH7m against virulent NDV, as well as against highly pathogenic avian influenza virus (HPAIV), was evaluated in specific-pathogen-free chickens. After a single immunization, all chickens developed NDV-specific, as well as AIV H7-specific, antibodies and were completely protected from clinical disease after infection with a lethal dose of virulent NDV or the homologous H7N1 HPAIV, while all control animals died within four days. Shedding of AIV challenge virus was strongly reduced compared to nonvaccinated control birds. Furthermore, the immunized birds developed antibodies against the AIV nucleoprotein after challenge infection. Thus, NDVH7m could be used as a marker vaccine against subtype H7 avian influenza.

The polygalacturonase gene BcMF2 from Brassica campestris is associated with intine development

PubMed Central

Huang, Li; Cao, Jiashu; Zhang, Aihong; Ye, Yiqun; Zhang, Yuchao; Liu, Tingting

2009-01-01

Brassica campestris Male Fertility 2 (BcMF2) is a putative polygalacturonase (PG) gene previously isolated from the flower bud of Chinese cabbage (Brassica campestris L. ssp. chinensis Makino, syn. B. rapa ssp. chinensis). This gene was found to be expressed specifically in tapetum and pollen after the tetrad stage of anther development. Antisense RNA technology was used to study the function of BcMF2 in Chinese cabbage. Scanning and transmission electron microscopy revealed that there were deformities in the transgenic mature pollen grains such as abnormal location of germinal furrows. In addition, the homogeneous pectic exintine layer facing the exterior seemed to be overdeveloped and predominantly occupied the intine, thus reversing the normal proportional distribution of the internal endintine layer and the external exintine layer. Since it is a continuation of the intine layer, the pollen tube wall could not grow normally. This resulted in the formation of a balloon-like swelling structure in the pollen tube tip in nearly 80% of the transgenic pollen grains. Premature degradation of tapetum was also found in these transgenic plants, which displayed decreased expression of the BcMF2 gene. BcMF2 might therefore encode a new PG with an important role in pollen wall development, possibly via regulation of pectin's dynamic metabolism. PMID:19039102

Characterization of myosin light chain in shrimp hemocytic phagocytosis.

PubMed

Han, Fang; Wang, Zhiyong; Wang, Xiaoqing

2010-11-01

Myosin light chain, a well-known cytoskeleton gene, regulates multiple processes that are involved in material transport, muscle shrink and cell division. However, its function in phagocytosis against invading pathogens in crustacean remains unknown. In this investigation, a myosin light chain gene was obtained from Marsupenaeus japonicus shrimp. The full-length cDNA of this gene was of 766 bp and an open reading frame (ORF) of 462 bp encoding a polypeptide of 153 amino acids. The myosin light chain protein was expressed in Escherichia coli and purified. Subsequently the specific antibody was raised using the purified GST fusion protein. As revealed by immuno-electron microscopy, the myosin light chain protein was only expressed in the dark bands of muscle. In the present study, the myosin light chain gene was up-regulated in the WSSV-resistant shrimp as revealed by real-time PCR and western blot. And the phagocytic percentage and phagocytic index using FITC-labeled Vibrio parahemolyticus were remarkably increased in the WSSV-resistant shrimp, suggesting that the myosin light chain protein was essential in hemocytic phagocytosis. On the other hand, RNAi assays indicated that the phagocytic percentage and phagocytic index were significantly decreased when the myosin light chain gene was silenced by sequence-specific siRNA. These findings suggested that myosin light chain protein was involved in the regulation of hemocytic phagocytosis of shrimp. Copyright 2010 Elsevier Ltd. All rights reserved.

Investigating the underlying mechanism of Saccharomyces cerevisiae in response to ethanol stress employing RNA-seq analysis.

PubMed

Li, Ruoyun; Xiong, Guotong; Yuan, Shukun; Wu, Zufang; Miao, Yingjie; Weng, Peifang

2017-11-03

Saccharomyces cerevisiae has been widely used for wine fermentation and bio-fuels production. A S. cerevisiae strain Sc131 isolated from tropical fruit shows good fermentation properties and ethanol tolerance, exhibiting significant potential in Chinese bayberry wine fermentation. In this study, RNA-sequence and RT-qPCR was used to investigate the transcriptome profile of Sc131 in response to ethanol stress. Scanning Electron Microscopy were carried out to observe surface morphology of yeast cells. Totally, 937 genes were identified differential expressed, including 587 up-regulated and 350 down-regulated genes, after 4-h ethanol stress (10% v/v). Transcriptomic analysis revealed that, most genes involved in regulating filamentous growth or pseudohyphal growth were significantly up-regulated in response to ethanol stress. The complex protein quality control machineries, Hsp90/Hsp70 and Hsp104/Hsp70/Hsp40 based chaperone system combining with ubiquitin-proteasome proteolytic pathway were both activated to recognize and degrade misfolding proteins. Genes related to biosynthesis and metabolism of two well-known stress-responsive substances trehalose and ergosterol were generally up-regulated, while genes associated with amino acids biosynthesis and metabolism processes were differentially expressed. Moreover, thiamine was also important in response to ethanol stress. This research may promote the potential applications of Sc131 in the fermentation of Chinese bayberry wine.

Single-molecule quantification of lipotoxic expression of activating transcription factor 3

PubMed Central

Wilson, Dennis W.; Rutledge, John C.

2014-01-01

Activating transcription factor 3 (ATF3) is a member of the mammalian activation transcription factor/cAMP, physiologically important in the regulation of pro- and anti-inflammatory target genes. We compared the induction of ATF3 protein as measured by Western blot analysis with single-molecule localization microscopy dSTORM to quantify the dynamics of accumulation of intranuclear ATF3 of triglyceride-rich (TGRL) lipolysis product-treated HAEC (Human Aortic Endothelial Cells). The ATF3 expression rate within the first three hours after treatment with TGRL lipolysis products is about 3500/h. After three hours we detected 33,090 ± 3,491 single-molecule localizations of ATF3. This was accompanied by significant structural changes in the F-actin network of the cells at ~3-fold increased localization precision compared to widefield microscopy after treatment. Additionally, we discovered a cluster size of approximately 384 nanometers of ATF3 molecules. We show for the first time the time course of ATF3 accumulation in the nucleus undergoing lipotoxic injury. Furthermore, we demonstrate ATF3 accumulation associated with increased concentrations of TGRL lipolysis products occurs in large aggregates. PMID:25189785

[Inhibition analysis of resveratrol against Vibrio parahaemolyticus biofilm based on RNA-Seq technology].

PubMed

Zhang, Fang; Zhu, Junli; Feng, Lifang

2016-05-04

Food phytochemicals as biofilm inhibitor of pathogens have been highlighted. Our study aimed to investigate the effects of resveratrol on biofilm formation of an aquatic pathogen Vibrio parahaemolyticus, and to elucidate the important regulatory genes. In the subinhibitory concentrations, the inhibition of resveratrol aganist biofilm and exopolysaccharides of V. parahaemolyticus was detected, and the differentially expressed genes were analyzed based on RNA-Seq. Four genes involved in biofilm formation was validated by qRT-PCR. The minimum inhibitory concentration of resveratrol against V. parahaemolyticus was 20 μg/mL. Resveratrol at the subinhibitory concentration of 5 μg/mL and 10 μg/mL significantly decreased the biofilm development and exopolysaccharides production in V. parahaemolyticus (P < 0.05). Scanning electron microscopy micrographs showed a significant reduction of adherence and extracellular polymeric substances. RNA-Seq analysis revealed that 22.6% up-regulated and 77.4% down-regulated gene (P < 0.05) after treatment by 10 μg/mL resveratrol among 106 differential gene expressions. These differential genes in V. parahaemolyticu focused on 7 metabolic pathways, and 14 genes involved in biofilm formation were down-regulated by resveratrol, such as outer membrane protein (W, YedS, OmpK), quorum sensing (LuxS), flagellin (FlaA), fimbrial assembly protein (PilQ), hemolysin secreted protein. qRT-PCR confirmed that the expressions of luxS, trh, tlh and flaA, was significantly repressed in the presence of resveratrol, which was consistent with transcriptomics data. Inhibitory activity of resveratrol on biofilm formation was assicated with multiple genes and diverse cellular processes in V. parahaemolyticus. These findings suggest that resveratrol would disturb various metabolic pathways, particularly quorum sensing system, adhesion process and membrane proteins secretion, resulting in inhibition of attachment and biofilm development. The present work provided valuable information to explore molecular mechanism of resveratrol as an novel anti-biofilm compound.

Analysis of the APETALA3- and PISTILLATA-like genes in Hedyosmum orientale (Chloranthaceae) provides insight into the evolution of the floral homeotic B-function in angiosperms

PubMed Central

Liu, Shujun; Sun, Yonghua; Du, Xiaoqiu; Xu, Qijiang; Wu, Feng; Meng, Zheng

2013-01-01

Background and Aims According to the floral ABC model, B-function genes appear to play a key role in the origin and diversification of the perianth during the evolution of angiosperms. The basal angiosperm Hedyosmum orientale (Chloranthaceae) has unisexual inflorescences associated with a seemingly primitive reproductive morphology and a reduced perianth structure in female flowers. The aim of this study was to investigate the nature of the perianth and the evolutionary state of the B-function programme in this species. Methods A series of experiments were conducted to characterize B-gene homologues isolated from H. orientale, including scanning electron microscopy to observe the development of floral organs, phylogenetic analysis to reconstruct gene evolutionary history, reverse transcription–PCR, quantitative real-time PCR and in situ hybridization to identify gene expression patterns, the yeast two-hybrid assay to explore protein dimerization affinities, and transgenic analyses in Arabidopsis thaliana to determine activities of the encoded proteins. Key Results The expression of HoAP3 genes was restricted to stamens, whereas HoPI genes were broadly expressed in all floral organs. HoAP3 was able to partially restore the stamen but not petal identity in Arabidopsis ap3-3 mutants. In contrast, HoPI could rescue aspects of both stamen and petal development in Arabidopsis pi-1 mutants. When the complete C-terminal sequence of HoPI was deleted, however, no or weak transgenic phenotypes were observed and homodimerization capability was completely abolished. Conclusions The results suggest that Hedyosmum AP3-like genes have an ancestral function in specifying male reproductive organs, and that the activity of the encoded PI-like proteins is highly conserved between Hedyosmum and Arabidopsis. Moreover, there is evidence that the C-terminal region is important for the function of HoPI. Our findings indicate that the development of the proposed perianth in Hedyosmum does not rely on the B homeotic function. PMID:23956161

A novel start codon mutation of the MERTK gene in a patient with retinitis pigmentosa

PubMed Central

Jinda, Worapoj; Poungvarin, Naravat; Taylor, Todd D.; Suzuki, Yutaka; Thongnoppakhun, Wanna; Limwongse, Chanin; Lertrit, Patcharee; Suriyaphol, Prapat

2016-01-01

Purpose Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous group of inherited retinal degenerations characterized by progressive loss of photoreceptor cells and RPE functions. More than 70 causative genes are known to be responsible for RP. This study aimed to identify the causative gene in a patient from a consanguineous family with childhood-onset severe retinal dystrophy. Methods To identify the defective gene, whole exome sequencing was performed. Candidate causative variants were selected and validated using Sanger sequencing. Segregation analysis of the causative gene was performed in additional family members. To verify that the mutation has an effect on protein synthesis, an expression vector containing the first ten amino acids of the mutant protein fused with the DsRed2 fluorescent protein was constructed and transfected into HEK293T cells. Expression of the fusion protein in the transfected cells was measured using fluorescence microscopy. Results By filtering against public variant databases, a novel homozygous missense mutation (c.3G>A) localized in the start codon of the MERTK gene was detected as a potentially pathogenic mutation for autosomal recessive RP. The c.3G>A mutation cosegregated with the disease phenotype in the family. No expression of the first ten amino acids of the MerTK mutant fused with the DsRed2 fluorescent protein was detected in HEK293T cells, indicating that the mutation affects the translation initiation site of the gene that may lead to loss of function of the MerTK signaling pathway. Conclusions We report a novel missense mutation (c.3G>A, p.0?) in the MERTK gene that causes severe vision impairment in a patient. Taken together with previous reports, our results expand the spectrum of MERTK mutations and extend our understanding of the role of the MerTK protein in the pathogenesis of retinitis pigmentosa. PMID:27122965

Single-molecule FRET reveals the energy landscape of the full-length SAM-I riboswitch.

PubMed

Manz, Christoph; Kobitski, Andrei Yu; Samanta, Ayan; Keller, Bettina G; Jäschke, Andres; Nienhaus, G Ulrich

2017-11-01

S-adenosyl-L-methionine (SAM) ligand binding induces major structural changes in SAM-I riboswitches, through which gene expression is regulated via transcription termination. Little is known about the conformations and motions governing the function of the full-length Bacillus subtilis yitJ SAM-I riboswitch. Therefore, we have explored its conformational energy landscape as a function of Mg 2+ and SAM ligand concentrations using single-molecule Förster resonance energy transfer (smFRET) microscopy and hidden Markov modeling analysis. We resolved four conformational states both in the presence and the absence of SAM and determined their Mg 2+ -dependent fractional populations and conformational dynamics, including state lifetimes, interconversion rate coefficients and equilibration timescales. Riboswitches with terminator and antiterminator folds coexist, and SAM binding only gradually shifts the populations toward terminator states. We observed a pronounced acceleration of conformational transitions upon SAM binding, which may be crucial for off-switching during the brief decision window before expression of the downstream gene.

Trastuzumab Alters the Expression of Genes Essential for Cardiac Function and Induces Ultrastructural Changes of Cardiomyocytes in Mice

PubMed Central

ElZarrad, M. Khair; Mukhopadhyay, Partha; Mohan, Nishant; Hao, Enkui; Dokmanovic, Milos; Hirsch, Dianne S.; Shen, Yi; Pacher, Pal; Wu, Wen Jin

2013-01-01

Treatment with trastuzumab, a humanized monoclonal antibody directed against the extracellular domain of Human Epidermal Growth Factor Receptor 2 (HER2), very successfully improves outcomes for women with HER2-positive breast cancer. However, trastuzumab treatment was recently linked to potentially irreversible serious cardiotoxicity, the mechanisms of which are largely elusive. This study reports that trastuzumab significantly alters the expression of myocardial genes essential for DNA repair, cardiac and mitochondrial functions, which is associated with impaired left ventricular performance in mice coupled with significant ultrastructural alterations in cardiomyocytes revealed by electron microscopy. Furthermore, trastuzumab treatment also promotes oxidative stress and apoptosis in myocardium of mice, and elevates serum levels of cardiac troponin-I (cTnI) and cardiac myosin light chain-1 (cMLC1). The elevated serum levels of cMLC1 in mice treated with trastuzumab highlights the potential that cMLC1 could be a useful biomarker for trastuzumab-induced cardiotoxicity. PMID:24255707

Schistosoma mansoni miracidia transformed by particle bombardment infect Biomphalaria glabrata snails and develop into transgenic sporocysts.

PubMed

Heyers, Oliver; Walduck, Anna K; Brindley, Paul J; Bleiss, Wilfrid; Lucius, Richard; Dorbic, Tomislav; Wittig, Burghardt; Kalinna, Bernd H

2003-10-01

Miracidia (and adults) of Schistosoma mansoni which had been subjected to particle bombardment with a plasmid DNA encoding enhanced green fluorescent protein (EGFP) under control of the S. mansoni heat shock protein 70 (HSP70) promoter and termination elements were shown to express the reporter gene. Bombarded miracidia were able to penetrate and establish in Biomphalaria glabrata the intermediate host snail. Gold particles could be detected in the germ balls of parasites in paraffin-sections of snail tissue. The bombarded miracidia were able to develop normally and to transform into mother sporocysts. Reporter gene activity could be determined at 10 days post-infection by RT-PCR in snail tissues, but not by microscopy or Western blot which probably reflected sub-optimal expression levels of constructs. Our findings indicated that it is feasible to return transgenic miracidia to the life cycle, a crucial step for the establishment of a transgenesis system for schistosomes.

Effects of Phonation Time and Magnitude Dose on Vocal Fold Epithelial Genes, Barrier Integrity, and Function

PubMed Central

Kojima, Tsuyoshi; Valenzuela, Carla V.; Novaleski, Carolyn K.; Van Deusen, Mark; Mitchell, Joshua R.; Garrett, C. Gaelyn; Sivasankar, M. Preeti; Rousseau, Bernard

2014-01-01

Objective To investigate the effects of increasing time and magnitude doses of vibration exposure on transcription of the vocal fold's junctional proteins, structural alterations, and functional tissue outcomes. Study Design Animal study. Methods 100 New Zealand White breeder rabbits were studied. Dependent variables were measured in response to increasing time doses (30, 60, or 120 minutes) and magnitude doses (control, modal intensity, and raised intensity) of vibration exposure. Messenger RNA expression of occludin, zonula occluden-1 (ZO-1), E-cadherin, β-catenin, interleukin 1β (IL-1β), cyclooxygenase-2 (COX-2), transforming growth factor β-1 (TGFβ1), and fibronectin were measured. Tissue structural alterations were assessed using transmission electron microscopy (TEM). Transepithelial resistance was used to measure functional tissue outcomes. Results Occludin gene expression was downregulated in vocal folds exposed to 120 minute time doses of raised intensity phonation, relative to control, and modal intensity phonation. ZO-1 gene expression was upregulated following a 120 minute time dose of modal intensity phonation, compared to control, and downregulated after a 120 minute time dose of raised intensity phonation, compared to modal intensity phonation. E-cadherin gene expression was downregulated after a120 minute time dose of raised intensity phonation, compared to control and modal intensity phonation. TEM revealed extensive desquamation of the stratified squamous epithelial cells with increasing time and magnitude doses of vibration exposure. A general observation of lower transepithelial resistance measures was made in tissues exposed to raised intensity phonation, compared to all other groups. Conclusions This study provides evidence of vocal fold tissue responses to varying time and magnitude doses of vibration exposure. Level of Evidence N/A PMID:25073715

Flower Development and Sex Determination between Male and Female Flowers in Vernicia fordii

PubMed Central

Mao, Yingji; Liu, Wenbo; Chen, Xue; Xu, Yang; Lu, Weili; Hou, Jinyan; Ni, Jun; Wang, Yuting; Wu, Lifang

2017-01-01

Vernicia fordii is a monoecious and diclinous species with male and female flowers on the same inflorescence. Low female to male flower ratio is one of the main reasons for low yield in this species. However, little is known of its floral development and sex determination. Here, according to the results of scanning electron microscopy and histological analysis, the floral development of V. fordii was divided into 12 stages and the first morphological divergence between the male and female flowers was found to occur at stage 7. The male flowers are always unisexual, but the female flowers present bisexual characteristics, with sterile stamen (staminode) restricted to pre-meiosis of mother sporogenous cells and cell death occurring at later development stages. To further elucidate the molecular mechanism underling sex determination at the divergence stage for male and female flowers, comparative transcriptome analysis was performed. In total, 56,065 unigenes were generated and 608 genes were differentially expressed between male and female flowers, among which 310 and 298 DEGs (differentially expressed genes) showed high expression levels in males and females, respectively. The transcriptome data showed that the sexual dimorphism of female flowers was affected by jasmonic acid, transcription factors, and some genes related to the floral meristem activity. Ten candidate genes showed consistent expression in the qRT-PCR validation and DEGs data. In this study, we provide developmental characterization and transcriptomic information for better understanding of the development of unisexual flowers and the regulatory networks underlying the mechanism of sex determination in V. fordii, which would be helpful in the molecular breeding of V. fordii to improve the yield output. PMID:28775735

Flower Development and Sex Determination between Male and Female Flowers in Vernicia fordii.

PubMed

Mao, Yingji; Liu, Wenbo; Chen, Xue; Xu, Yang; Lu, Weili; Hou, Jinyan; Ni, Jun; Wang, Yuting; Wu, Lifang

2017-01-01

Vernicia fordii is a monoecious and diclinous species with male and female flowers on the same inflorescence. Low female to male flower ratio is one of the main reasons for low yield in this species. However, little is known of its floral development and sex determination. Here, according to the results of scanning electron microscopy and histological analysis, the floral development of V. fordii was divided into 12 stages and the first morphological divergence between the male and female flowers was found to occur at stage 7. The male flowers are always unisexual, but the female flowers present bisexual characteristics, with sterile stamen (staminode) restricted to pre-meiosis of mother sporogenous cells and cell death occurring at later development stages. To further elucidate the molecular mechanism underling sex determination at the divergence stage for male and female flowers, comparative transcriptome analysis was performed. In total, 56,065 unigenes were generated and 608 genes were differentially expressed between male and female flowers, among which 310 and 298 DEGs (differentially expressed genes) showed high expression levels in males and females, respectively. The transcriptome data showed that the sexual dimorphism of female flowers was affected by jasmonic acid, transcription factors, and some genes related to the floral meristem activity. Ten candidate genes showed consistent expression in the qRT-PCR validation and DEGs data. In this study, we provide developmental characterization and transcriptomic information for better understanding of the development of unisexual flowers and the regulatory networks underlying the mechanism of sex determination in V. fordii , which would be helpful in the molecular breeding of V. fordii to improve the yield output.

Rgs13 constrains early B cell responses and limits germinal center sizes.

PubMed

Hwang, Il-Young; Hwang, Kyung-Sun; Park, Chung; Harrison, Kathleen A; Kehrl, John H

2013-01-01

Germinal centers (GCs) are microanatomic structures that develop in secondary lymphoid organs in response to antigenic stimulation. Within GCs B cells clonally expand and their immunoglobulin genes undergo class switch recombination and somatic hypermutation. Transcriptional profiling has identified a number of genes that are prominently expressed in GC B cells. Among them is Rgs13, which encodes an RGS protein with a dual function. Its canonical function is to accelerate the intrinsic GTPase activity of heterotrimeric G-protein α subunits at the plasma membrane, thereby limiting heterotrimeric G-protein signaling. A unique, non-canonical function of RGS13 occurs following translocation to the nucleus, where it represses CREB transcriptional activity. The functional role of RGS13 in GC B cells is unknown. To create a surrogate marker for Rgs13 expression and a loss of function mutation, we inserted a GFP coding region into the Rgs13 genomic locus. Following immunization GFP expression rapidly increased in activated B cells, persisted in GC B cells, but declined in newly generated memory B and plasma cells. Intravital microscopy of the inguinal lymph node (LN) of immunized mice revealed the rapid appearance of GFP(+) cells at LN interfollicular regions and along the T/B cell borders, and eventually within GCs. Analysis of WT, knock-in, and mixed chimeric mice indicated that RGS13 constrains extra-follicular plasma cell generation, GC size, and GC B cell numbers. Analysis of select cell cycle and GC specific genes disclosed an aberrant gene expression profile in the Rgs13 deficient GC B cells. These results indicate that RGS13, likely acting at cell membranes and in nuclei, helps coordinate key decision points during the expansion and differentiation of naive B cells.

Identification and Characterization of Two Temperature-Induced Surface-Associated Proteins of Streptococcus suis with High Homologies to Members of the Arginine Deiminase System of Streptococcus pyogenes

PubMed Central

Winterhoff, Nora; Goethe, Ralph; Gruening, Petra; Rohde, Manfred; Kalisz, Henryk; Smith, Hilde E.; Valentin-Weigand, Peter

2002-01-01

The present study was performed to identify stress-induced putative virulence proteins of Streptococcus suis. For this, protein expression patterns of streptococci grown at 32, 37, and 42°C were compared by one- and two-dimensional gel electrophoresis. Temperature shifts from 32 and 37 to 42°C induced expression of two cell wall-associated proteins with apparent molecular masses of approximately 47 and 53 kDa. Amino-terminal sequence analysis of the two proteins indicated homologies of the 47-kDa protein with an ornithine carbamoyltransferase (OCT) from Streptococcus pyogenes and of the 53-kDa protein with the streptococcal acid glycoprotein (SAGP) from S. pyogenes, an arginine deiminase (AD) recently proposed as a putative virulence factor. Cloning and sequencing the genes encoding the putative OCT and AD of S. suis, octS and adiS, respectively, revealed that they had 81.2 (octS) and 80.2% (adiS) identity with the respective genes of S. pyogenes. Both genes belong to the AD system, also found in other bacteria. Southern hybridization analysis demonstrated the presence of the adiS gene in all 42 serotype 2 and 9 S. suis strains tested. In 9 of these 42 strains, selected randomly, we confirmed expression of the AdiS protein, homologous to SAGP, by immunoblot analysis using a specific antiserum against the SAGP of S. pyogenes. In all strains AD activity was detected. Furthermore, by immunoelectron microscopy using the anti-S. pyogenes SAGP antiserum we were able to demonstrate that the AdiS protein is expressed on the streptococcal surface in association with the capsular polysaccharides but is not coexpressed with them. PMID:12446626

Immunological and molecular polymorphisms of OspC, an immunodominant major outer surface protein of Borrelia burgdorferi.

PubMed Central

Wilske, B; Preac-Mursic, V; Jauris, S; Hofmann, A; Pradel, I; Soutschek, E; Schwab, E; Will, G; Wanner, G

1993-01-01

The gene of the immunodominant major protein pC of Borrelia burgdorferi was previously cloned and sequenced (R. Fuchs, S. Jauris, F. Lottspeich, V. Preac-Mursic, B. Wilske, and E. Soutschek, Mol. Microbiol. 6:503-509, 1992). pC is abundantly expressed on the outer surface of B. burgdorferi, as demonstrated by immunoelectron microscopy with monoclonal antibody L22 1F8. Accordingly, pC is renamed OspC, by analogy to the outer surface proteins OspA and OspB. Western immunoblot analysis of 45 B. burgdorferi isolates with monoclonal antibodies revealed that OspC is immunologically heterogeneous. Partial sequence analysis of the ospC gene confirmed the protein heterogeneity at the genetic level. We found that the degree of identity between the ospC partial sequences of five strains representing different OspA serotypes was only 63.3 to 85.4%. Immunological heterogeneity was also observed among representatives of the three newly designated genospecies of B. burgdorferi sensu lato, B. burgdorferi sensu stricto, B. garinii, and group VS461. Heterogeneity was confirmed for B. garinii at the genetic level. The ospC gene was also cloned from strains that did not express OspC, and antibody-reactive OspC was expressed in Escherichia coli. In addition, OspC-expressing variants were obtained from a nonexpressing strain by plating single colonies on solid medium. These findings confirm that the ospC gene is also present in nonexpressing strains. Because OspC is an immunodominant protein for the early immune response in Lyme borreliosis and was effective as a vaccine in an animal model, the immunological and molecular polymorphisms of ospC and OspC have important implications for the development of diagnostic reagents and vaccines. Images PMID:8478108

A comparative study of the effects of retinol and retinoic acid on histological, molecular, and clinical properties of human skin.

PubMed

Kong, Rong; Cui, Yilei; Fisher, Gary J; Wang, Xiaojuan; Chen, Yinbei; Schneider, Louise M; Majmudar, Gopa

2016-03-01

All-trans retinol, a precursor of retinoic acid, is an effective anti-aging treatment widely used in skin care products. In comparison, topical retinoic acid is believed to provide even greater anti-aging effects; however, there is limited research directly comparing the effects of retinol and retinoic acid on skin. In this study, we compare the effects of retinol and retinoic acid on skin structure and expression of skin function-related genes and proteins. We also examine the effect of retinol treatment on skin appearance. Skin histology was examined by H&E staining and in vivo confocal microscopy. Expression levels of skin genes and proteins were analyzed using RT-PCR and immunohistochemistry. The efficacy of a retinol formulation in improving skin appearance was assessed using digital image-based wrinkle analysis. Four weeks of retinoic acid and retinol treatments both increased epidermal thickness, and upregulated genes for collagen type 1 (COL1A1), and collagen type 3 (COL3A1) with corresponding increases in procollagen I and procollagen III protein expression. Facial image analysis showed a significant reduction in facial wrinkles following 12 weeks of retinol application. The results of this study demonstrate that topical application of retinol significantly affects both cellular and molecular properties of the epidermis and dermis, as shown by skin biopsy and noninvasive imaging analyses. Although the magnitude tends to be smaller, retinol induces similar changes in skin histology, and gene and protein expression as compared to retinoic acid application. These results were confirmed by the significant facial anti-aging effect observed in the retinol efficacy clinical study. © 2015 Wiley Periodicals, Inc.

High-purity flow sorting of early meiocytes based on DNA analysis of guinea pig spermatogenic cells.

PubMed

Rodríguez-Casuriaga, Rosana; Geisinger, Adriana; Santiñaque, Federico F; López-Carro, Beatriz; Folle, Gustavo A

2011-08-01

Mammalian spermatogenesis is still nowadays poorly understood at the molecular level. Testis cellular heterogeneity is a major drawback for spermatogenic gene expression studies, especially when research is focused on stages that are usually very short and poorly represented at the cellular level such as initial meiotic prophase I (i.e., leptotene [L] and zygotene [Z]). Presumably, genes whose products are involved in critical meiotic events such as alignment, pairing and recombination of homologous chromosomes are expressed during the short stages of early meiotic prophase. Aiming to characterize mammalian early meiotic gene expression, we have found the guinea pig (Cavia porcellus) as an especially attractive model. A detailed analysis of its first spermatogenic wave by flow cytometry (FCM) and optical microscopy showed that guinea pig testes exhibit a higher representation of early meiotic stages compared to other studied rodents, partly because of their longer span, and also as a result of the increased number of cells entering meiosis. Moreover, we have found that adult guinea pig testes exhibit a peculiar 4C DNA content profile, with a bimodal peak for L/Z and P spermatocytes that is absent in other rodents. Besides, we show that this unusual 4C peak allows the separation by FCM of highly pure L/Z spermatocyte populations aside from pachytene ones, even from adult individuals. To our knowledge, this is the first report on an accurate and suitable method for highly pure early meiotic prophase cell isolation from adult mammals, and thus sets an interesting approach for gene expression studies aiming at a deeper understanding of the molecular groundwork underlying male gamete production. Copyright © 2011 International Society for Advancement of Cytometry.

Characterization of susceptibility and resistance responses to potato cyst nematode (Globodera spp.) infection of tomato lines in the absence and presence of the broad-spectrum nematode resistance Hero gene.

PubMed

Sobczak, Miroslaw; Avrova, Anna; Jupowicz, Justyna; Phillips, Mark S; Ernst, Karin; Kumar, Amar

2005-02-01

The tomato Hero A gene is the only member of a multigene family that confers a high level (>80%) of resistance to all the economically important pathotypes of potato cyst nematode (PCN) species Globodera rostochiensis and G. pallida. Although the resistance levels of transgenic tomato lines were similar to those of the tomato line LA1792 containing the introgressed Hero multigene family, transgenic potato plants expressing the tomato Hero A gene are not resistant to PCNs. Comparative microscopy studies of in vitro infected roots of PCN-susceptible tomato cv. Money Maker, the resistant breeding line LA1792, and transgenic line L10 with Ro1 pathotype have revealed no statistically significant difference in the number of juveniles invading roots. However, syncytia (specialized feeding cells) induced in LA1792 and L10 roots mostly were found to have degenerated a few days after their induction, and a few surviving syncytia were able to support only the development of males rather than females. Thus, the ratio between males and females was biased towards males on LA1792 and L10 roots. A series of changes occur in resistant plants leading to formation of a layer of necrotic cells separating the syncytium from stellar conductive tissues and this is followed by degradation of the syncytium. Although the Hero A gene is expressed in all tissues, including roots, stems, leaves, and flower buds, its expression is upregulated in roots in response to PCN infection. Moreover, the expression profiles of the Hero A correlates with the timing of death of the syncytium.

Targeted gene delivery to the synovial pannus in antigen-induced arthritis by ultrasound-targeted microbubble destruction in vivo.

PubMed

Xiang, Xi; Tang, Yuanjiao; Leng, Qianying; Zhang, Lingyan; Qiu, Li

2016-02-01

The purpose of this study was to optimize an ultrasound-targeted microbubble destruction (UTMD) technique to improve the in vivo transfection efficiency of the gene encoding enhanced green fluorescent protein (EGFP) in the synovial pannus in an antigen-induced arthritis rabbit model. A mixture of microbubbles and plasmids was locally injected into the knee joints of an antigen-induced arthritis (AIA) rabbits. The plasmid concentrations and ultrasound conditions were varied in the experiments. We also tested local articular and intravenous injections. The rabbits were divided into five groups: (1) ultrasound+microbubbles+plasmid; (2) ultrasound+plasmid; (3) microbubble+plasmid; (4) plasmid only; (5) untreated controls. EGFP expression was observed by fluorescent microscope and immunohistochemical staining in the synovial pannus of each group. The optimal plasmid dosage and ultrasound parameter were determined based on the results of EGFP expression and the present and absent of tissue damage under light microscopy. The irradiation procedure was performed to observe the duration of the EGFP expression in the synovial pannus and other tissues and organs, as well as the damage to the normal cells. The optimal condition was determined to be a 1-MHz ultrasound pulse applied for 5 min with a power output of 2 W/cm(2) and a 20% duty cycle along with 300 μg of plasmid. Under these conditions, the synovial pannus showed significant EGFP expression without significant damage to the surrounding normal tissue. The EGFP expression induced by the local intra-articular injection was significantly more increased than that induced by the intravenous injection. The EGFP expression in the synovial pannus of the ultrasound+microbubbles+plasmid group was significantly higher than that of the other four groups (P<0.05). The expression peaked on day 5, remained detectable on day 40 and disappeared on day 60. No EGFP expression was detected in the other tissues and organs. The UTMD technique can significantly enhance the in vivo gene transfection efficiency without significant tissue damage in the synovial pannus of an AIA model. Thus, this could become a safe and effective non-viral gene transfection procedure for arthritis therapy. Copyright © 2015 Elsevier B.V. All rights reserved.

Simultaneous visualization of the subfemtomolar expression of microRNA and microRNA target gene using HILO microscopy.

PubMed

Lin, Yi-Zhen; Ou, Da-Liang; Chang, Hsin-Yuan; Lin, Wei-Yu; Hsu, Chiun; Chang, Po-Ling

2017-09-01

The family of microRNAs (miRNAs) not only plays an important role in gene regulation but is also useful for the diagnosis of diseases. A reliable method with high sensitivity may allow researchers to detect slight fluctuations in ultra-trace amounts of miRNA. In this study, we propose a sensitive imaging method for the direct probing of miR-10b (miR-10b-3p, also called miR-10b*) and its target ( HOXD10 mRNA) in fixed cells based on the specific recognition of molecular beacons combined with highly inclined and laminated optical sheet (HILO) fluorescence microscopy. The designed dye-quencher-labelled molecular beacons offer excellent efficiencies of fluorescence resonance energy transfer that allow us to detect miRNA and the target mRNA simultaneously in hepatocellular carcinoma cells using HILO fluorescence microscopy. Not only can the basal trace amount of miRNA be observed in each individual cell, but the obtained images also indicate that this method is useful for monitoring the fluctuations in ultra-trace amounts of miRNA when the cells are transfected with a miRNA precursor or a miRNA inhibitor (anti-miR). Furthermore, a reasonable causal relation between the miR-10b and HOXD10 expression levels was observed in miR-10b* precursor-transfected cells and miR-10b* inhibitor-transfected cells. The trends of the miRNA alterations obtained using HILO microscopy completely matched the RT-qPCR data and showed remarkable reproducibility (the coefficient of variation [CV] = 0.86%) and sensitivity (<1.0 fM). This proposed imaging method appears to be useful for the simultaneous visualisation of ultra-trace amounts of miRNA and target mRNA and excludes the procedures for RNA extraction and amplification. Therefore, the visualisation of miRNA and the target mRNA should facilitate the exploration of the functions of ultra-trace amounts of miRNA in fixed cells in biological studies and may serve as a powerful tool for diagnoses based on circulating cancer cells.

Segmentation and detection of fluorescent 3D spots.

PubMed

Ram, Sundaresh; Rodríguez, Jeffrey J; Bosco, Giovanni

2012-03-01

The 3D spatial organization of genes and other genetic elements within the nucleus is important for regulating gene expression. Understanding how this spatial organization is established and maintained throughout the life of a cell is key to elucidating the many layers of gene regulation. Quantitative methods for studying nuclear organization will lead to insights into the molecular mechanisms that maintain gene organization as well as serve as diagnostic tools for pathologies caused by loss of nuclear structure. However, biologists currently lack automated and high throughput methods for quantitative and qualitative global analysis of 3D gene organization. In this study, we use confocal microscopy and fluorescence in-situ hybridization (FISH) as a cytogenetic technique to detect and localize the presence of specific DNA sequences in 3D. FISH uses probes that bind to specific targeted locations on the chromosomes, appearing as fluorescent spots in 3D images obtained using fluorescence microscopy. In this article, we propose an automated algorithm for segmentation and detection of 3D FISH spots. The algorithm is divided into two stages: spot segmentation and spot detection. Spot segmentation consists of 3D anisotropic smoothing to reduce the effect of noise, top-hat filtering, and intensity thresholding, followed by 3D region-growing. Spot detection uses a Bayesian classifier with spot features such as volume, average intensity, texture, and contrast to detect and classify the segmented spots as either true or false spots. Quantitative assessment of the proposed algorithm demonstrates improved segmentation and detection accuracy compared to other techniques. Copyright © 2012 International Society for Advancement of Cytometry.

In Vivo Study of Trichoderma-Pathogen-Plant Interactions, Using Constitutive and Inducible Green Fluorescent Protein Reporter Systems

PubMed Central

Lu, Zexun; Tombolini, Riccardo; Woo, Sheridan; Zeilinger, Susanne; Lorito, Matteo; Jansson, Janet K.

2004-01-01

Plant tissue colonization by Trichoderma atroviride plays a critical role in the reduction of diseases caused by phytopathogenic fungi, but this process has not been thoroughly studied in situ. We monitored in situ interactions between gfp-tagged biocontrol strains of T. atroviride and soilborne plant pathogens that were grown in cocultures and on cucumber seeds by confocal scanning laser microscopy and fluorescence stereomicroscopy. Spores of T. atroviride adhered to Pythium ultimum mycelia in coculture experiments. In mycoparasitic interactions of T. atroviride with P. ultimum or Rhizoctonia solani, the mycoparasitic hyphae grew alongside the pathogen mycelia, and this was followed by coiling and formation of specialized structures similar to hooks, appressoria, and papillae. The morphological changes observed depended on the pathogen tested. Branching of T. atroviride mycelium appeared to be an active response to the presence of the pathogenic host. Mycoparasitism of P. ultimum by T. atroviride occurred on cucumber seed surfaces while the seeds were germinating. The interaction of these fungi on the cucumber seeds was similar to the interaction observed in coculture experiments. Green fluorescent protein expression under the control of host-inducible promoters was also studied. The induction of specific Trichoderma genes was monitored visually in cocultures, on plant surfaces, and in soil in the presence of colloidal chitin or Rhizoctonia by confocal microscopy and fluorescence stereomicroscopy. These tools allowed initiation of the mycoparasitic gene expression cascade to be monitored in vivo. PMID:15128569

Apoptosis Governs the Elimination of Schistosoma japonicum from the Non-Permissive Host Microtus fortis

PubMed Central

Peng, Jinbiao; Gobert, Geoffrey N.; Hong, Yang; Jiang, Weibin; Han, Hongxiao; McManus, Donald P.; Wang, Xinzhi; Liu, Jinming; Fu, Zhiqiang; Shi, Yaojun; Lin, Jiaojiao

2011-01-01

The reed vole, Microtus fortis, is the only known mammalian host in which schistosomes of Schistosoma japonicum are unable to mature and cause significant pathogenesis. However, little is known about how Schistosoma japonicum maturation (and, therefore, the development of schistosomiasis) is prevented in M. fortis. In the present study, the ultrastructure of 10 days post infection schistosomula from BALB/c mice and M. fortis were first compared using scanning electron microscopy and transmission electron microscopy. Electron microscopic investigations showed growth retardation and ultrastructural differences in the tegument and sub-tegumental tissues as well as in the parenchymal cells of schistosomula from M. fortis compared with those in BALB/c mice. Then, microarray analysis revealed significant differential expression between the schistosomula from the two rodents, with 3,293 down-regulated (by ≥2-fold) and 71 up-regulated (≥2 fold) genes in schistosomula from the former. The up-regulated genes included a proliferation-related gene encoding granulin (Grn) and tropomyosin. Genes that were down-regulated in schistosomula from M. fortis included apoptosis-inhibited genes encoding a baculoviral IAP repeat-containing protein (SjIAP) and cytokine-induced apoptosis inhibitor (SjCIAP), genes encoding molecules involved in insulin metabolism, long-chain fatty acid metabolism, signal transduction, the transforming growth factor (TGF) pathway, the Wnt pathway and in development. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) and PI/Annexin V-FITC assays, caspase 3/7 activity analysis, and flow cytometry revealed that the percentages of early apoptotic and late apoptotic and/or necrotic cells, as well as the level of caspase activity, in schistosomula from M. fortis were all significantly higher than in those from BALB/c mice. PMID:21731652

AUXIN RESPONSE FACTOR17 Is Essential for Pollen Wall Pattern Formation in Arabidopsis1[C][W][OA

PubMed Central

Yang, Jun; Tian, Lei; Sun, Ming-Xi; Huang, Xue-Yong; Zhu, Jun; Guan, Yue-Feng; Jia, Qi-Shi; Yang, Zhong-Nan

2013-01-01

In angiosperms, pollen wall pattern formation is determined by primexine deposition on the microspores. Here, we show that AUXIN RESPONSE FACTOR17 (ARF17) is essential for primexine formation and pollen development in Arabidopsis (Arabidopsis thaliana). The arf17 mutant exhibited a male-sterile phenotype with normal vegetative growth. ARF17 was expressed in microsporocytes and microgametophytes from meiosis to the bicellular microspore stage. Transmission electron microscopy analysis showed that primexine was absent in the arf17 mutant, which leads to pollen wall-patterning defects and pollen degradation. Callose deposition was also significantly reduced in the arf17 mutant, and the expression of CALLOSE SYNTHASE5 (CalS5), the major gene for callose biosynthesis, was approximately 10% that of the wild type. Chromatin immunoprecipitation and electrophoretic mobility shift assays showed that ARF17 can directly bind to the CalS5 promoter. As indicated by the expression of DR5-driven green fluorescent protein, which is an synthetic auxin response reporter, auxin signaling appeared to be specifically impaired in arf17 anthers. Taken together, our results suggest that ARF17 is essential for pollen wall patterning in Arabidopsis by modulating primexine formation at least partially through direct regulation of CalS5 gene expression. PMID:23580594

Gene within gene configuration and expression of the Drosophila melanogaster genes lethal(2) neighbour of tid [l(2)not] and lethal(2) relative of tid[l(2)rot].

PubMed

Kurzik-Dumke, U; Kaymer, M; Gundacker, D; Debes, A; Labitzke, K

1997-10-24

In this paper, we describe the structure and temporal expression pattern of the Drosophila melanogaster genes l(2)not and l(2)rot located at locus 59F5 vis à vis the tumor suppressor gene l(2)tid described previously and exhibiting a gene within gene configuration. The l(2)not protein coding region, 1530 nt, is divided into two exons by an intron, 2645 nt, harboring the genes l(2)rot, co-transcribed from the same DNA strand, and l(2)tid, co-transcribed from the opposite DNA strand, located vis à vis. To determine proteins encoded by the genes described in this study polyclonal rabbit antibodies (Ab), anti-Not and anti-Rot, were generated. Immunostaining of developmental Western blots with the anti-Not Ab resulted in the identification of a 45-kDa protein, Not45, which is smaller than the Not56 protein predicted from the sequence. Its localization in endoplasmic reticulum (ER) was established by immunoelectron microscopy of Drosophila melanogaster Schneider 2 cells. Not45 shows significant homology to yeast ALG3 protein acting as a dolichol mannosyltransferase in the asparagine-linked glycosylation. It is synthesized ubiquitously throughout embryonic life. The protein predicted from the l(2)rot sequence, Rot57, shows a homology to the NS2B protein of the yellow fever virus1 (yefv1). The results of l(2)rot RNA analysis by developmental Northern blot and by in situ RNA localization, as well as the results of the protein analysis via Western blot and immunohistochemistry suggest that l(2)rot is transcribed but not translated. Since RNAs encoded by the genes l(2)tid and l(2)rot are complementary and l(2)rot is presumably not translated we performed preliminary experiments on the function of the l(2)rot RNA as a natural antisense RNA (asRNA) regulator of l(2)tid expression, expressed in the same temporal and spatial manner as the l(2)tid- and l(2)not RNA. l(2)tid knock-out by antisense RNA yielded late embryonic lethality resulting from multiple morphogenetic defects.

Identification of Sporopollenin as the Outer Layer of Cell Wall in Microalga Chlorella protothecoides.

PubMed

He, Xi; Dai, Junbiao; Wu, Qingyu

2016-01-01

Chlorella protothecoides has been put forth as a promising candidate for commercial biodiesel production. However, the cost of biodiesel remains much higher than diesel from fossil fuel sources, partially due to the high costs of oil extraction from algae. Here, we identified the presence of a sporopollenin layer outside the polysaccharide cell wall; this was evaluated using transmission electron microscopy, 2-aminoethanol treatment, acetolysis, and Fourier Transform Infrared Spectroscopy. We also performed bioinformatics analysis of the genes of the C. protothecoides genome that are likely involved in sporopollenin synthesis, secretion, and translocation, and evaluated the expression of these genes via real-time PCR. We also found that that removal of this sporopollenin layer greatly improved the efficiency of oil extraction.

Single molecule fluorescence microscopy for ultra-sensitive RNA expression profiling

NASA Astrophysics Data System (ADS)

Hesse, Jan; Jacak, Jaroslaw; Regl, Gerhard; Eichberger, Thomas; Aberger, Fritz; Schlapak, Robert; Howorka, Stefan; Muresan, Leila; Frischauf, Anna-Maria; Schütz, Gerhard J.

2007-02-01

We developed a microarray analysis platform for ultra-sensitive RNA expression profiling of minute samples. It utilizes a novel scanning system for single molecule fluorescence detection on cm2 size samples in combination with specialized biochips, optimized for low autofluorescence and weak unspecific adsorption. 20 μg total RNA was extracted from 10 6 cells of a human keratinocyte cell line (HaCaT) and reversely transcribed in the presence of Alexa647-aha-dUTP. 1% of the resulting labeled cDNA was used for complex hybridization to a custom-made oligonucleotide microarray representing a set of 125 different genes. For low abundant genes, individual cDNA molecules hybridized to the microarray spots could be resolved. Single cDNA molecules hybridized to the chip surface appeared as diffraction limited features in the fluorescence images. The à trous wavelet method was utilized for localization and counting of the separated cDNA signals. Subsequently, the degree of labeling of the localized cDNA molecules was determined by brightness analysis for the different genes. Variations by factors up to 6 were found, which in conventional microarray analysis would result in a misrepresentation of the relative abundance of mRNAs.

Sub-Inhibitory Concentrations of Trans-Cinnamaldehyde Attenuate Virulence in Cronobacter sakazakii in Vitro

PubMed Central

Amalaradjou, Mary Anne Roshni; Kim, Kwang Sik; Venkitanarayanan, Kumar

2014-01-01

Cronobacter sakazakii is a foodborne pathogen, which causes a life-threatening form of meningitis, necrotizing colitis and meningoencephalitis in neonates and children. Epidemiological studies implicate dried infant formula as the principal source of C. sakazakii. In this study, we investigated the efficacy of sub-inhibitory concentrations (SIC) of trans-cinnamaldehyde (TC), an ingredient in cinnamon, for reducing C. sakazakii virulence in vitro using cell culture, microscopy and gene expression assays. TC significantly (p ≤ 0.05) suppressed C. sakazakii adhesion to and invasion of human and rat intestinal epithelial cells, and human brain microvascular endothelial cells. In addition, TC inhibited C. sakazakii survival and replication in human macrophages. We also observed that TC reduced the ability of C. sakazakii to cause cell death in rat intestinal cells, by inhibiting nitric oxide production. Results from gene expression studies revealed that TC significantly downregulated the virulence genes critical for motility, host tissue adhesion and invasion, macrophage survival, and LPS (Lipopolysaccharide) synthesis in C. sakazakii. The efficacy of TC in attenuating these major virulence factors in C. sakazakii underscores its potential use in the prevention and/or control of infection caused by this pathogen. PMID:24837831

Sub-inhibitory concentrations of trans-cinnamaldehyde attenuate virulence in Cronobacter sakazakii in vitro.

PubMed

Amalaradjou, Mary Anne Roshni; Kim, Kwang Sik; Venkitanarayanan, Kumar

2014-05-15

Cronobacter sakazakii is a foodborne pathogen, which causes a life-threatening form of meningitis, necrotizing colitis and meningoencephalitis in neonates and children. Epidemiological studies implicate dried infant formula as the principal source of C. sakazakii. In this study, we investigated the efficacy of sub-inhibitory concentrations (SIC) of trans-cinnamaldehyde (TC), an ingredient in cinnamon, for reducing C. sakazakii virulence in vitro using cell culture, microscopy and gene expression assays. TC significantly (p ≤ 0.05) suppressed C. sakazakii adhesion to and invasion of human and rat intestinal epithelial cells, and human brain microvascular endothelial cells. In addition, TC inhibited C. sakazakii survival and replication in human macrophages. We also observed that TC reduced the ability of C. sakazakii to cause cell death in rat intestinal cells, by inhibiting nitric oxide production. Results from gene expression studies revealed that TC significantly downregulated the virulence genes critical for motility, host tissue adhesion and invasion, macrophage survival, and LPS (Lipopolysaccharide) synthesis in C. sakazakii. The efficacy of TC in attenuating these major virulence factors in C. sakazakii underscores its potential use in the prevention and/or control of infection caused by this pathogen.

Colocalization of kindlin-1, kindlin-2, and migfilin at keratinocyte focal adhesion and relevance to the pathophysiology of Kindler syndrome.

PubMed

Lai-Cheong, J E; Ussar, S; Arita, K; Hart, I R; McGrath, J A

2008-09-01

Kindler syndrome (KS) results from pathogenic loss-of-function mutations in the KIND1 gene, which encodes kindlin-1, a focal adhesion and actin cytoskeleton-related protein. How and why abnormalities in kindlin-1 disrupt keratinocyte cell biology in KS, however, is not yet known. In this study, we identified two previously unreported binding proteins of kindlin-1: kindlin-2 and migfilin. Co-immunoprecipitation and confocal microscopy studies show that these three proteins bind to each other and colocalize at focal adhesion in HaCaT cells and normal human keratinocytes. Moreover, loss-of-function mutations in KIND1 result in marked variability in kindlin-1 immunolabeling in KS skin, which is mirrored by similar changes in kindlin-2 and migfilin immunoreactivity. Kindlin-1, however, may function independently of kindlin-2 and migfilin, as loss of kindlin-1 expression in HaCaT keratinocytes by RNA interference and in KS keratinocytes does not affect KIND2 or FBLIM1 (migfilin) gene expression or kindlin-2 and migfilin protein localization. In addition to identifying protein-binding partners for kindlin-1, this study also highlights that KIND1 gene expression and kindlin-1 protein labeling are not always reduced in KS, findings that are relevant to the accurate laboratory diagnosis of this genodermatosis by skin immunohistochemistry.

Syringic Acid Extracted from Herba dendrobii Prevents Diabetic Cataract Pathogenesis by Inhibiting Aldose Reductase Activity

PubMed Central

Wei, Xiaoyong; Chen, Dan; Yi, Yanchun; Qi, Hui; Gao, Xinxin; Fang, Hua; Gu, Qiong; Wang, Ling; Gu, Lianquan

2012-01-01

Objective. Effects of Syringic acid (SA) extracted from dendrobii on diabetic cataract (DC) pathogenesis were explored. Methods. Both in vitro and in vivo DC lens models were established using D-gal, and proliferation of HLEC exposed to SA was determined by MMT assay. After 60-day treatment with SA, rat lens transparency was observed by anatomical microscopy using a slit lamp. SA protein targets were extracted and isolated using 2-DE and MALDI TOF/TOF. AR gene expression was investigated using qRT-PCR. Interaction sites and binding characteristics were determined by molecule-docking techniques and dynamic models. Results. Targeting AR, SA provided protection from D-gal-induced damage by consistently maintaining lens transparency and delaying lens turbidity development. Inhibition of AR gene expression by SA was confirmed by qRT-PCR. IC50 of SA for inhibition of AR activity was 213.17 μg/mL. AR-SA binding sites were Trp111, His110, Tyr48, Trp20, Trp79, Leu300, and Phe122. The main binding modes involved hydrophobic interactions and hydrogen bonding. The stoichiometric ratio of non-covalent bonding between SA and AR was 1.0 to 13.3. Conclusion. SA acts to prevent DC in rat lenses by inhibiting AR activity and gene expression, which has potential to be developed into a novel drug for therapeutic management of DC. PMID:23365598

An investigation into the effect of surface roughness of stainless steel on human umbilical vein endothelial cell gene expression.

PubMed

McLucas, E; Moran, M T; Rochev, Y; Carroll, W M; Smith, T J

2006-01-01

The surface properties of vascular devices dictate the initial postimplantation reactions that occur and thus the efficacy of the implantation procedure. Over the last number of years, a number of different stent designs have emerged and stents are generally polished to a mirror finish during the manufacturing procedure. This study sought to investigate the effect of stainless steel surface roughness on endothelial cell gene expression using an appropriate cell culture in vitro assay system. Stainless steel discs were roughened by shot blasting or polished by mechanical polishing. The surface roughness of the treated and untreated discs was determined by atomic force microscopy (AFM). Cells were seeded on collagen type 1 gels and left to attach for 24 h. Stainless steel discs of varying roughness were then placed in contact with the cells and incubated for 24 h. RNA extractions and quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was then performed to determine the expression levels of candidate genes in the treated cells compared to suitable control cells. E-selectin and vascular cellular adhesion molecule (VCAM-1) were found to be significantly up-regulated in cells incubated with polished and roughened samples, indicating endothelial cell activation and inflammation. This study indicates that the surface roughness of stainless steel is an important surface property in the development of vascular stents.

Testosterone-induced modulation of peroxisomal morphology and peroxisome-related gene expression in brown trout (Salmo trutta f. fario) primary hepatocytes.

PubMed

Lopes, Célia; Malhão, Fernanda; Guimarães, Cláudia; Pinheiro, Ivone; Gonçalves, José F; Castro, L Filipe C; Rocha, Eduardo; Madureira, Tânia V

2017-12-01

Disruption of androgenic signaling has been linked to possible cross-modulation with other hormone-mediated pathways. Therefore, our objective was to explore effects caused by testosterone - T (1, 10 and 50μM) in peroxisomal signaling of brown trout hepatocytes. To study the underlying paths involved, several co-exposure conditions were tested, with flutamide - F (anti-androgen) and ICI 182,780 - ICI (anti-estrogen). Molecular and morphological approaches were both evaluated. Peroxisome proliferator-activated receptor alpha (PPARα), catalase and urate oxidase were the selected targets for gene expression analysis. The vitellogenin A gene was also included as a biomarker of estrogenicity. Peroxisome relative volumes were estimated by immunofluorescence, and transmission electron microscopy was used for qualitative morphological control. The single exposures of T caused a significant down-regulation of urate oxidase (10 and 50μM) and a general up-regulation of vitellogenin. A significant reduction of peroxisome relative volumes and smaller peroxisome profiles were observed at 50μM. Co-administration of T and ICI reversed the morphological modifications and vitellogenin levels. The simultaneous exposure of T and F caused a significant and concentration-dependent diminishing in vitellogenin expression. Together, the findings suggest that in the tested model, T acted via both androgen and estrogen receptors to shape the peroxisomal related targets. Copyright © 2017 Elsevier B.V. All rights reserved.

Duck hepatitis A virus structural proteins expressed in insect cells self-assemble into virus-like particles with strong immunogenicity in ducklings.

PubMed

Wang, Anping; Gu, Lingling; Wu, Shuang; Zhu, Shanyuan

2018-02-01

Duck hepatitis A virus (DHAV), a non-enveloped ssRNA virus, can cause a highly contagious disease in young ducklings. The three capsid proteins of VP0, VP1 and VP3 are translated within a single large open reading frame (ORF) and hydrolyzed by protease 3CD. However, little is known on whether the recombinant viral structural proteins (VPs) expressed in insect cells could spontaneously assemble into virus-like particles (VLPs) and whether these VLPs could induce protective immunity in young ducklings. To address these issues, the structural polyprotein precursor gene P1 and the protease gene 3CD were amplified by PCR, and the recombinant proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures and immunogenicity. The recombinant proteins expressed in Sf9 cells were detected by indirect immunofluorescence assay and Western blot analysis. Electron microscopy showed that the recombinant proteins spontaneously assembled into VLPs in insect cells. Western blot analysis of the purified VLPs revealed that the VLPs were composed with the three structural proteins. In addition, vaccination with the VLPs induced high humoral immune response and provided strong protection. Therefore, our findings may provide a framework for development of new vaccines for the prevention of duck viral hepatitis. Copyright © 2018 Elsevier B.V. All rights reserved.

Regulation of vascular endothelial growth factor-C by tumor necrosis factor-α in the conjunctiva and pterygium.

PubMed

Dong, Yoko; Kase, Satoru; Dong, Zhenyu; Fukuhara, Junichi; Tagawa, Yoshiaki; Ishizuka, Erdal Tan; Murata, Miyuki; Shinmei, Yasuhiro; Ohguchi, Takeshi; Kanda, Atsuhiro; Noda, Kousuke; Ishida, Susumu

2016-08-01

Vascular endothelial growth factor C (VEGF-C) plays an important role in the development of a pterygium through lymphangiogenesis. We examined the association between VEGF-C and tumor necrosis factor-α (TNF-α) in the pathogenesis of pterygia. Cultured conjunctival epithelial cells were treated with TNF-α, and the gene expression levels of VEGFC were evaluated by quantitative polymerase chain reaction (qPCR) and VEGF-C protein expression levels were measured using an enzyme-linked immunosorbent assay (ELISA). In addition, using ELISA, we evaluated the VEGF-C protein expression in the supernatants of cultured conjunctival epithelial cells, in which we neutralized TNF-α using anti‑TNF-α antibody. The gene expression of tumor necrosis factor receptor superfamily, member 1A (TNFRSF1A), known as TNF receptor 1 (TNFR1), was confirmed using reverse transcription PCR in cultured conjunctival epithelial cells. Immunofluorescence microscopy was used to examine the localization of VEGF-C and TNFR1 in pterygium tissues and TNFR1 expression in cultured conjunctival epithelial cells. Immunohistochemistry was used to examine the localization of TNFR1 in pterygia and normal conjunctival tissues. VEGFC gene expression increased in cultured conjunctival epithelial cells 24 h after the addition of TNF-α. The secretion of VEGF-C protein was significantly increased 48 h after the stimulation of cultured conjunctival epithelial cells with TNF-α. Increased VEGF-C protein secretion stimulated by TNF-α was significantly reduced by anti-TNF-α neutralizing antibody treatment. In cultured conjunctival epithelial cells, TNFRSF1A and TNFR1 were expressed. TNFR1 was immunolocalized in normal conjunctival tissues and in human pterygium tissues as well as in VEGF‑C‑positive epithelial cells from human pterygia. Our data demonstrate that TNF-α mediates VEGF-C expression, which plays a critical role in the pathogenesis of pterygia.

Effect of Eicosapentaenoic Acid and Docosahexaenoic Acid on Myogenesis and Mitochondrial Biosynthesis during Murine Skeletal Muscle Cell Differentiation.

PubMed

Hsueh, Tun-Yun; Baum, Jamie I; Huang, Yan

2018-01-01

Polyunsaturated fatty acids are important nutrients for human health, especially omega-3 fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which have been found to play positive roles in the prevention of various diseases. However, previous studies have reported that excessive omega-3 fatty acids supplement during pregnancy caused side effects such as slower neural transmission times and postnatal growth restriction. In this study, we investigated the effect of EPA and DHA on mitochondrial function and gene expression in C2C12 myoblasts during skeletal muscle differentiation. C2C12 myoblasts were cultured to confluency and then treated with differentiation medium that contained fatty acids (50-µM EPA and DHA). After 72 h of myogenic differentiation, mRNA was collected, and gene expression was analyzed by real-time PCR. Microscopy was used to examine cell morphology following treatment with fatty acids. The effect of EPA and DHA on cellular oxygen consumption was measured using a Seahorse XF24 Analyzer. Cells treated with fatty acids had fewer myotubes formed ( P ≤ 0.05) compared with control cells. The expression of the genes related to myogenesis was significantly lower ( P ≤ 0.05) in cells treated with fatty acids, compared with control cells. Genes associated with adipogenesis had higher ( P ≤ 0.05) expression after treatment with fatty acids. Also, the mitochondrial biogenesis decreased with lower ( P ≤ 0.05) gene expression and lower ( P ≤ 0.05) mtDNA/nDNA ratio in cells treated with fatty acids compared with control cells. However, the expression of genes related to peroxisome biosynthesis was higher ( P ≤ 0.05) in cells treated with fatty acids. Moreover, fatty-acid treatment reduced ( P ≤ 0.05) oxygen consumption rate under oligomycin-inhibited (reflecting proton leak) and uncoupled conditions. Our data imply that fatty acids might reduce myogenesis and increase adipogenesis in myotube formation. Fatty acids may also decrease cell metabolism by reducing mitochondrial biogenesis as well as respiration rate. This study suggests that the maternal overdosage of EPA and DHA may influence fetal muscle development, increase intramuscular adipose tissue deposition in offspring, and have a long-term effect on the development of metabolic diseases such as obesity and diabetes in adult offspring.

Effect of Eicosapentaenoic Acid and Docosahexaenoic Acid on Myogenesis and Mitochondrial Biosynthesis during Murine Skeletal Muscle Cell Differentiation

PubMed Central

Hsueh, Tun-Yun; Baum, Jamie I.; Huang, Yan

2018-01-01

Polyunsaturated fatty acids are important nutrients for human health, especially omega-3 fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which have been found to play positive roles in the prevention of various diseases. However, previous studies have reported that excessive omega-3 fatty acids supplement during pregnancy caused side effects such as slower neural transmission times and postnatal growth restriction. In this study, we investigated the effect of EPA and DHA on mitochondrial function and gene expression in C2C12 myoblasts during skeletal muscle differentiation. C2C12 myoblasts were cultured to confluency and then treated with differentiation medium that contained fatty acids (50-µM EPA and DHA). After 72 h of myogenic differentiation, mRNA was collected, and gene expression was analyzed by real-time PCR. Microscopy was used to examine cell morphology following treatment with fatty acids. The effect of EPA and DHA on cellular oxygen consumption was measured using a Seahorse XF24 Analyzer. Cells treated with fatty acids had fewer myotubes formed (P ≤ 0.05) compared with control cells. The expression of the genes related to myogenesis was significantly lower (P ≤ 0.05) in cells treated with fatty acids, compared with control cells. Genes associated with adipogenesis had higher (P ≤ 0.05) expression after treatment with fatty acids. Also, the mitochondrial biogenesis decreased with lower (P ≤ 0.05) gene expression and lower (P ≤ 0.05) mtDNA/nDNA ratio in cells treated with fatty acids compared with control cells. However, the expression of genes related to peroxisome biosynthesis was higher (P ≤ 0.05) in cells treated with fatty acids. Moreover, fatty-acid treatment reduced (P ≤ 0.05) oxygen consumption rate under oligomycin-inhibited (reflecting proton leak) and uncoupled conditions. Our data imply that fatty acids might reduce myogenesis and increase adipogenesis in myotube formation. Fatty acids may also decrease cell metabolism by reducing mitochondrial biogenesis as well as respiration rate. This study suggests that the maternal overdosage of EPA and DHA may influence fetal muscle development, increase intramuscular adipose tissue deposition in offspring, and have a long-term effect on the development of metabolic diseases such as obesity and diabetes in adult offspring. PMID:29594127

Products of three accessory genes, pilB, pilC, and pilD, are required for biogenesis of Pseudomonas aeruginosa pili.

PubMed Central

Nunn, D; Bergman, S; Lory, S

1990-01-01

The polar pili of Pseudomonas aeruginosa are composed of monomers of the pilin structural subunits. The biogenesis of pili involves the synthesis of pilin precursor, cleavage of a six-amino-acid leader peptide, membrane translocation, and assembly of monomers into a filamentous structure extending from the bacterial surface. This report describes three novel genes necessary for the formation of pili. DNA sequences adjacent to pilA, the pilin structural gene, were cloned and mutagenized with transposon Tn5. Each of the insertions were introduced into the chromosome of P. aeruginosa PAK by gene replacement. The effect of the Tn5 insertions in the bacterial chromosome on pilus assembly was assessed by electron microscopy and sensitivity of mutants to a pilus-specific bacteriophage. The resultant mutants were also tested for synthesis and membrane localization of the pilin antigen in order to define the genes required for maturation, export, and assembly of pilin. A 4.0-kilobase-pair region of DNA adjacent to the pilin structural gene was found to be essential for formation of pili. This region was sequenced and found to contain three open reading frames coding for 62-, 38- to 45-, and 28- to 32-kilodalton proteins (pilB, pilC, and pilD, respectively). Three proteins of similar molecular weight were expressed in Escherichia coli from the 4.0-kilobase-pair fragment flanking pilA with use of a T7 promoter-polymerase expression system. The results of the analyses of the three genes and the implications for pilin assembly and maturation are discussed. Images PMID:1971619

Transcriptional and microscopic analyses of citrus stem and root responses to Candidatus Liberibacter asiaticus infection.

PubMed

Aritua, Valente; Achor, Diann; Gmitter, Frederick G; Albrigo, Gene; Wang, Nian

2013-01-01

Huanglongbing (HLB) is the most destructive disease that affects citrus worldwide. The disease has been associated with Candidatus Liberibacter. HLB diseased citrus plants develop a multitude of symptoms including zinc and copper deficiencies, blotchy mottle, corky veins, stunting, and twig dieback. Ca. L. asiaticus infection also seriously affects the roots. Previous study focused on gene expression of leaves and fruit to Ca. L. asiaticus infection. In this study, we compared the gene expression levels of stems and roots of healthy plants with those in Ca. L. asiaticus infected plants using microarrays. Affymetrix microarray analysis showed a total of 988 genes were significantly altered in expression, of which 885 were in the stems, and 111 in the roots. Of these, 551 and 56 were up-regulated, while 334 and 55 were down-regulated in the stem and root samples of HLB diseased trees compared to healthy plants, respectively. Dramatic differences in the transcriptional responses were observed between citrus stems and roots to Ca. L. asiaticus infection, with only 8 genes affected in both the roots and stems. The affected genes are involved in diverse cellular functions, including carbohydrate metabolism, cell wall biogenesis, biotic and abiotic stress responses, signaling and transcriptional factors, transportation, cell organization, protein modification and degradation, development, hormone signaling, metal handling, and redox. Microscopy analysis showed the depletion of starch in the roots of the infected plants but not in healthy plants. Collapse and thickening of cell walls were observed in HLB affected roots, but not as severe as in the stems. This study provides insight into the host response of the stems and roots to Ca. L. asiaticus infection.

Transcriptional and Microscopic Analyses of Citrus Stem and Root Responses to Candidatus Liberibacter asiaticus Infection

PubMed Central

Aritua, Valente; Achor, Diann; Gmitter, Frederick G.; Albrigo, Gene; Wang, Nian

2013-01-01

Huanglongbing (HLB) is the most destructive disease that affects citrus worldwide. The disease has been associated with Candidatus Liberibacter. HLB diseased citrus plants develop a multitude of symptoms including zinc and copper deficiencies, blotchy mottle, corky veins, stunting, and twig dieback. Ca. L. asiaticus infection also seriously affects the roots. Previous study focused on gene expression of leaves and fruit to Ca. L. asiaticus infection. In this study, we compared the gene expression levels of stems and roots of healthy plants with those in Ca. L. asiaticus infected plants using microarrays. Affymetrix microarray analysis showed a total of 988 genes were significantly altered in expression, of which 885 were in the stems, and 111 in the roots. Of these, 551 and 56 were up-regulated, while 334 and 55 were down-regulated in the stem and root samples of HLB diseased trees compared to healthy plants, respectively. Dramatic differences in the transcriptional responses were observed between citrus stems and roots to Ca. L. asiaticus infection, with only 8 genes affected in both the roots and stems. The affected genes are involved in diverse cellular functions, including carbohydrate metabolism, cell wall biogenesis, biotic and abiotic stress responses, signaling and transcriptional factors, transportation, cell organization, protein modification and degradation, development, hormone signaling, metal handling, and redox. Microscopy analysis showed the depletion of starch in the roots of the infected plants but not in healthy plants. Collapse and thickening of cell walls were observed in HLB affected roots, but not as severe as in the stems. This study provides insight into the host response of the stems and roots to Ca. L. asiaticus infection. PMID:24058486

Effects of zoledronic acid and geranylgeraniol on the cellular behaviour and gene expression of primary human alveolar osteoblasts.

PubMed

Zafar, S; Coates, D E; Cullinan, M P; Drummond, B K; Milne, T; Seymour, G J

2016-11-01

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a serious complication of bisphosphonate therapy. The mechanism underlying BRONJ pathogenesis is poorly understood. To determine the effects of zoledronic acid (ZA) and geranylgeraniol (GGOH) on the mevalonate pathway (MVP) in osteoblasts generated from the human mandibular alveolar bone in terms of cell viability/proliferation, migration, apoptosis and gene expression. Primary human osteoblasts (HOBs) isolated from the mandibular alveolar bone were phenotyped. HOBs were cultured with or without ZA and GGOH for up to 72 h. Cellular behaviour was examined using a CellTiter-Blue® viability assay, an Ibidi culture-insert migration assay, an Apo-ONE® Homogeneous Caspase-3/7 apoptosis assay and transmission electron microscopy (TEM). Quantitative real-time reverse transcriptase polymerase chain reaction (qRT 2 -PCR) was used to determine the simultaneous expression of 168 osteogenic and angiogenic genes modulated in the presence of ZA and GGOH. ZA decreased cell viability and migration and induced apoptosis in HOBs. TEM revealed signs of apoptosis in ZA-treated HOBs. However, the co-addition of GGOH ameliorated the effect of ZA and partially restored the cells to the control state. Twenty-eight genes in the osteogenic array and 27 genes in the angiogenic array were significantly regulated in the presence of ZA compared with those in the controls at one or more time points. The cytotoxic effect of ZA on HOBs and its reversal by the addition of GGOH suggests that the effect of ZA on HOBs is mediated via the MVP. The results suggest that GGOH could be used as a possible therapeutic/preventive strategy for BRONJ.

MLF1 interacting protein: a potential gene therapy target for human prostate cancer?

PubMed

Zhang, Lei; Ji, Guoqing; Shao, Yuzhang; Qiao, Shaoyi; Jing, Yuming; Qin, Rongliang; Sun, Huiming; Shao, Chen

2015-02-01

Here, we investigated the role of one gene that has been previously associated with human prostate carcinoma cells-myelodysplasia/myeloid leukemia factor 1 interacting protein (MLF1IP)-in order to better ascertain its role in human prostate carcinogenesis. The prostate cancer cell line PC-3 was lentivirally transfected to silence endogenous MLF1IP gene expression, which was confirmed by real-time quantitative PCR (RT-qPCR). Cellomics ArrayScan VTI imaging and MTT assays were conducted to assess cell proliferation. Cell cycle phase arrest and apoptosis were assayed by flow cytometry. Colony formation was assessed by fluorescence microscopy. MLF1IP gene expression was also analyzed by RT-qPCR in sixteen prostate cancer tissue samples and six healthy control prostate tissue samples from human patients. Cell proliferation was significantly inhibited in MLF1IP-silenced cells relative to control cells. G1 phase, S and G2/M phase cell counts were not significantly changed in MLF1IP-silenced cells relative to control cells. Apoptosis was significantly increased in MLF1IP-silenced cells, while MLF1IP-silenced cells displayed a significantly reduced number of cell colonies, compared to control cells. The 16 human prostate cancer tissue samples revealed no clear upregulation or downregulation in MLF1IP gene expression. MLF1IP significantly promotes prostate cancer cell proliferation and colony formation and significantly inhibits apoptosis without affecting cell cycle phase arrest. Further study is required to conclusively determine whether MLF1IP is upregulated in human prostate cancer tumors and to determine the precise cellular mechanism(s) for MLF1IP in prostate carcinogenesis.

Tegumental Phosphodiesterase SmNPP-5 Is a Virulence Factor for Schistosomes ▿

PubMed Central

Bhardwaj, Rita; Krautz-Peterson, Greice; Da'dara, Akram; Tzipori, Saul; Skelly, Patrick J.

2011-01-01

The intravascular trematode Schistosoma mansoni is a causative agent of schistosomiasis, a disease that constitutes a major health problem globally. In this study we cloned and characterized the schistosome tegumental phosphodiesterase SmNPP-5 and evaluated its role in parasite virulence. SmNPP-5 is a 52.5-kDa protein whose gene is rapidly turned on in the intravascular parasitic life stages, following invasion of the definitive host. Highest expression is found in mated adult males. As revealed by immunofluorescence analysis, SmNPP-5 protein is found prominently in the dorsal surface of the tegument of males. Localization by immuno-electron microscopy illustrates a unique pattern of immunogold-labeled SmNPP-5 within the tegument; some immunogold particles are scattered throughout the tissue, but many are clustered in tight arrays. To determine the importance of the protein for the parasites, RNA interference (RNAi) was employed to knock down expression of the SmNPP-5-encoding gene in schistosomula and adult worms. Both quantitative real-time PCR (qRT-PCR) and Western blotting confirmed successful and robust gene suppression. In addition, the suppression and the ectolocalization of this enzyme in live parasites were evident because of a significantly impaired ability of the suppressed parasites to hydrolyze exogenously added phosphodiesterase substrate p-nitrophenyl 5′-dTMP (p-Nph-5′-TMP). The effects of suppressing expression of the SmNPP-5 gene in vivo were tested by injecting parasites into mice. It was found that, unlike controls, parasites whose SmNPP-5 gene was demonstrably suppressed at the time of host infection were greatly impaired in their ability to establish infection. These results demonstrate that SmNPP-5 is a virulence factor for schistosomes. PMID:21825060

BioShuttle-mediated Plasmid Transfer

PubMed Central

Braun, Klaus; von Brasch, Leonie; Pipkorn, Ruediger; Ehemann, Volker; Jenne, Juergen; Spring, Herbert; Debus, Juergen; Didinger, Bernd; Rittgen, Werner; Waldeck, Waldemar

2007-01-01

An efficient gene transfer into target tissues and cells is needed for safe and effective treatment of genetic diseases like cancer. In this paper, we describe the development of a transport system and show its ability for transporting plasmids. This non-viral peptide-based BioShuttle-mediated transfer system consists of a nuclear localization address sequence realizing the delivery of the plasmid phNIS-IRES-EGFP coding for two independent reporter genes into nuclei of HeLa cells. The quantification of the transfer efficiency was achieved by measurements of the sodium iodide symporter activity. EGFP gene expression was measured with Confocal Laser Scanning Microscopy and quantified with biostatistical methods by analysis of the frequency of the amplitude distribution in the CLSM images. The results demonstrate that the “BioShuttle”-Technology is an appropriate tool for an effective transfer of genetic material carried by a plasmid. PMID:18026568

Repressing a Repressor

PubMed Central

Silverstone, Aron L.; Jung, Hou-Sung; Dill, Alyssa; Kawaide, Hiroshi; Kamiya, Yuji; Sun, Tai-ping

2001-01-01

RGA (for repressor of ga1-3) and SPINDLY (SPY) are likely repressors of gibberellin (GA) signaling in Arabidopsis because the recessive rga and spy mutations partially suppressed the phenotype of the GA-deficient mutant ga1-3. We found that neither rga nor spy altered the GA levels in the wild-type or the ga1-3 background. However, expression of the GA biosynthetic gene GA4 was reduced 26% by the rga mutation, suggesting that partial derepression of the GA response pathway by rga resulted in the feedback inhibition of GA4 expression. The green fluorescent protein (GFP)–RGA fusion protein was localized to nuclei in transgenic Arabidopsis. This result supports the predicted function of RGA as a transcriptional regulator based on sequence analysis. Confocal microscopy and immunoblot analyses demonstrated that the levels of both the GFP-RGA fusion protein and endogenous RGA were reduced rapidly by GA treatment. Therefore, the GA signal appears to derepress the GA signaling pathway by degrading the repressor protein RGA. The effect of rga on GA4 gene expression and the effect of GA on RGA protein level allow us to identify part of the mechanism by which GA homeostasis is achieved. PMID:11449051

Production of chimeric recombinant single domain antibody-green fluorescent fusion protein in Chinese hamster ovary cells.

PubMed

Bazl, M Rajabi; Rasaee, M J; Foruzandeh, M; Rahimpour, A; Kiani, J; Rahbarizadeh, F; Alirezapour, B; Mohammadi, M

2007-02-01

There is an increasing interest in the application of nanobodies such as VHH in the field of therapy and imaging. In the present study a stable genetically engineered cell line of Chinese hamster ovary (CHO) origin transfected using two sets of expression vectors was constructed in order to permit the cytoplasmic and extracellular expression of single domain antibody along with green fluorescent protein (GFP) as reporter gene. The quality of the constructs were examined both by the restriction map as well as sequence analysis. The gene transfection and protein expression was further examined by reverse transcription-polymerase chain reaction (RT-PCR). The transfected cells were grown in 200 microg/mL hygromycin containing media and the stable cell line obtained showed fluorescent activity for more than a period of 180 days. The production of fusion protein was also detected by fluorescent microscopy, fluorescent spectroscopy as well as by enzyme-linked immunosorbent assay (ELISA) analysis. This strategy allows a rapid production of recombinant fluobodies involving VHH, which can be used in various experiments such as imaging and detection in which a primary labeled antibody is required.

Dorso-ventral skin characterization of the farmed fish gilthead seabream (Sparus aurata)

PubMed Central

Cordero, Héctor; Ceballos-Francisco, Diana; Cuesta, Alberto

2017-01-01

The skin is the first barrier of defence in fish, protecting against any external stressor and preserving the integrity and homeostasis of the fish body. The aim of this study was to characterise gilthead seabream skin by isolating cells and studying the cell cycle by flow cytometry, to study the skin histology by scanning electron microscopy and the transcription level of some immune-relevant genes by RT-PCR. Furthermore, the results obtained from samples taken from the dorsal and the ventral part of the specimens are compared. No differences were observed in the cell cycle of cells isolated from the dorsal and ventral zones of the skin or in the gene expression of the genes studied in both epidermal zones. However, the epidermis thickness of the ventral skin was higher than that of the dorsal skin, as demonstrated by image analysis using light microscopy. Besides, scanning electron microscopy pointed to a greater cell size and area of microridges in the apical part of the dorsal epidermal cells compared with ventral skin epidermal cells. This study represents a step forward in our knowledge of the skin structure of an important farmed teleost, gilthead seabream, one of the most commonly farmed fish worldwide. Furthermore, for functional characterization, experimental wounds were carried out comparing the wound healing rate between the dorsal and ventral regions of skin over the time. The results showed higher ratio of wound healing in the ventral region, whose wounds were closed after 15 days, compared to dorsal region of skin. Taking into account all together, this study represents a step forward in our knowledge of the skin structure and skin regeneration of an important farmed teleost, gilthead seabream, one of the most commonly farmed fish worldwide. PMID:28666033

Deciphering the Minimal Algorithm for Development and Information-genesis

NASA Astrophysics Data System (ADS)

Li, Zhiyuan; Tang, Chao; Li, Hao

During development, cells with identical genomes acquires different fates in a highly organized manner. In order to decipher the principles underlining development, we used C.elegans as the model organism. Based on a large set of microscopy imaging, we first constructed a ``standard worm'' in silico: from the single zygotic cell to about 500 cell stage, the lineage, position, cell-cell contact and gene expression dynamics are quantified for each cell in order to investigate principles underlining these intensive data. Next, we reverse-engineered the possible gene-gene/cell-cell interaction rules that are capable of running a dynamic model recapitulating the early fate decisions during C.elegans development. we further formulized the C.elegans embryogenesis in the language of information genesis. Analysis towards data and model uncovered the global landscape of development in the cell fate space, suggested possible gene regulatory architectures and cell signaling processes, revealed diversity and robustness as the essential trade-offs in development, and demonstrated general strategies in building multicellular organisms.

Knockdown of Host Antioxidant Defense Genes Enhances the Effect of Glucantime on Intracellular Leishmania braziliensis in Human Macrophages.

PubMed

Téllez, Jair; Romero, Ibeth; Soares, Maurilio José; Steindel, Mario; Romanha, Alvaro José

2017-07-01

Leishmaniasis is a neglected tropical disease that affects millions of people worldwide and represents a major public health problem. Information on protein expression patterns and functional roles within the context of Leishmania -infected human monocyte-derived macrophages (MDMs) under drug treatment conditions is essential for understanding the role of these cells in leishmaniasis treatment. We analyzed functional changes in the expression of human MDM genes and proteins during in vitro infection by Leishmania braziliensis and treatment with Glucantime (Sb V ), using quantitative PCR (qPCR) arrays, Western blotting, confocal microscopy, and small interfering RNA (siRNA) human gene inhibition assays. Comparison of the results from gene transcription and protein expression analyses revealed that glutathione S -transferase π1 (GSTP1), glutamate-cysteine ligase modifier subunit (GCLM), glutathione reductase (GSR), glutathione synthetase (GSS), thioredoxin (TRX), and ATP-binding cassette, subfamily B, member 5 (ABCB5), were strongly upregulated at both the mRNA and protein levels in human MDMs that were infected and treated, compared to the control group. Subcellular localization studies showed a primarily phagolysosomal location for the ABCB5 transporter, indicating that this protein may be involved in the transport of Sb V By inducing a decrease in L. braziliensis intracellular survival in THP-1 macrophages, siRNA silencing of GSTP1 , GSS , and ABCB5 resulted in an increased leishmanicidal effect of Sb V exposure in vitro Our results suggest that human MDMs infected with L. braziliensis and treated with Sb V express increased levels of genes participating in antioxidant defense, whereas our functional analyses provide evidence for the involvement of human MDMs in drug detoxification. Therefore, we conclude that GSS, GSTP1, and ABCB5 proteins represent potential targets for enhancing the leishmanicidal activity of Glucantime. Copyright © 2017 American Society for Microbiology.

Induced pluripotent stem cell models of Zellweger spectrum disorder show impaired peroxisome assembly and cell type-specific lipid abnormalities.

PubMed

Wang, Xiao-Ming; Yik, Wing Yan; Zhang, Peilin; Lu, Wange; Huang, Ning; Kim, Bo Ram; Shibata, Darryl; Zitting, Madison; Chow, Robert H; Moser, Ann B; Steinberg, Steven J; Hacia, Joseph G

2015-08-29

Zellweger spectrum disorder (PBD-ZSD) is a disease continuum caused by mutations in a subset of PEX genes required for normal peroxisome assembly and function. They highlight the importance of peroxisomes in the development and functions of the central nervous system, liver, and other organs. To date, the underlying bases for the cell-type specificity of disease are not fully elucidated. Primary skin fibroblasts from seven PBD-ZSD patients with biallelic PEX1, PEX10, PEX12, or PEX26 mutations and three healthy donors were transduced with retroviral vectors expressing Yamanaka reprogramming factors. Candidate induced pluripotent stem cells (iPSCs) were subject to global gene expression, DNA methylation, copy number variation, genotyping, in vitro differentiation and teratoma formation assays. Confirmed iPSCs were differentiated into neural progenitor cells (NPCs), neurons, oligodendrocyte precursor cells (OPCs), and hepatocyte-like cell cultures with peroxisome assembly evaluated by microscopy. Saturated very long chain fatty acid (sVLCFA) and plasmalogen levels were determined in primary fibroblasts and their derivatives. iPSCs were derived from seven PBD-ZSD patient-derived fibroblasts with mild to severe peroxisome assembly defects. Although patient and control skin fibroblasts had similar gene expression profiles, genes related to mitochondrial functions and organelle cross-talk were differentially expressed among corresponding iPSCs. Mitochondrial DNA levels were consistent among patient and control fibroblasts, but varied among all iPSCs. Relative to matching controls, sVLCFA levels were elevated in patient-derived fibroblasts, reduced in patient-derived iPSCs, and not significantly different in patient-derived NPCs. All cell types derived from donors with biallelic null mutations in a PEX gene showed plasmalogen deficiencies. Reporter gene assays compatible with high content screening (HCS) indicated patient-derived OPC and hepatocyte-like cell cultures had impaired peroxisome assembly. Normal peroxisome activity levels are not required for cellular reprogramming of skin fibroblasts. Patient iPSC gene expression profiles were consistent with hypotheses highlighting the role of altered mitochondrial activities and organelle cross-talk in PBD-ZSD pathogenesis. sVLCFA abnormalities dramatically differed among patient cell types, similar to observations made in iPSC models of X-linked adrenoleukodystrophy. We propose that iPSCs could assist investigations into the cell type-specificity of peroxisomal activities, toxicology studies, and in HCS for targeted therapies for peroxisome-related disorders.

Silver nanoparticles induce tight junction disruption and astrocyte neurotoxicity in a rat blood–brain barrier primary triple coculture model

PubMed Central

Xu, Liming; Dan, Mo; Shao, Anliang; Cheng, Xiang; Zhang, Cuiping; Yokel, Robert A; Takemura, Taro; Hanagata, Nobutaka; Niwa, Masami; Watanabe, Daisuke

2015-01-01

Background Silver nanoparticles (Ag-NPs) can enter the brain and induce neurotoxicity. However, the toxicity of Ag-NPs on the blood–brain barrier (BBB) and the underlying mechanism(s) of action on the BBB and the brain are not well understood. Method To investigate Ag-NP suspension (Ag-NPS)-induced toxicity, a triple coculture BBB model of rat brain microvascular endothelial cells, pericytes, and astrocytes was established. The BBB permeability and tight junction protein expression in response to Ag-NPS, NP-released Ag ions, and polystyrene-NP exposure were investigated. Ultrastructural changes of the microvascular endothelial cells, pericytes, and astrocytes were observed using transmission electron microscopy (TEM). Global gene expression of astrocytes was measured using a DNA microarray. Results A triple coculture BBB model of primary rat brain microvascular endothelial cells, pericytes, and astrocytes was established, with the transendothelial electrical resistance values >200 Ω·cm2. After Ag-NPS exposure for 24 hours, the BBB permeability was significantly increased and expression of the tight junction (TJ) protein ZO-1 was decreased. Discontinuous TJs were also observed between microvascular endothelial cells. After Ag-NPS exposure, severe mitochondrial shrinkage, vacuolations, endoplasmic reticulum expansion, and Ag-NPs were observed in astrocytes by TEM. Global gene expression analysis showed that three genes were upregulated and 20 genes were downregulated in astrocytes treated with Ag-NPS. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the 23 genes were associated with metabolic processes, biosynthetic processes, response to stimuli, cell death, the MAPK pathway, and so on. No GO term and KEGG pathways were changed in the released-ion or polystyrene-NP groups. Ag-NPS inhibited the antioxidant defense of the astrocytes by increasing thioredoxin interacting protein, which inhibits the Trx system, and decreasing Nr4a1 and Dusp1. Meanwhile, Ag-NPS induced inflammation and apoptosis through modulation of the MAPK pathway or B-cell lymphoma-2 expression or mTOR activity in astrocytes. Conclusion These results draw our attention to the importance of Ag-NP-induced toxicity on the neurovascular unit and provide a better understanding of its toxicological mechanisms on astrocytes. PMID:26491287

[Thrombopenia and radial aplasia: 2 cases with platelet function and ultrastructural studies of megakaryocytes and platelets (author's transl)].

PubMed

Juhan, I; Bayle, J; Mattei, J F; Thevenieau, D; Perrimond, H; Muratore, R

1979-10-01

The authors report on two cases of congenital thrombopenia with radial aplasia. Both children display several formative abnormalities and a mild thrombopenia; hemorragic manifestations occurred in the first case only. Megacryoblastic to platelets series, as studied with electronic microscopy, show small-sized, "microcytic" and hypogranular megacaryocytes, displaying a maturative disorder (dysmegacaryocytopoiesis). In functional studies, platelets of the first patient show an imperfect nucleotidic release and do not agregate normally with ristocetin. The second case exhibits mostly a PF3 reduction. The variety of expression of the megacaryocytic-platelets disorders appears likewise in the squelettal and visceral malformations. The whole disorder could be ascribed to a pleiotropic abnormal gene with a variable expressivity.

In vitro study for laser gene transfer in BHK-21 fibroblast cell line

NASA Astrophysics Data System (ADS)

Abdel Aziz, M.; Salem, D. S.; Salama, M. S.; Badr, Y.

2009-02-01

Modifications to our previously introduced system for laser microbeam cell surgery were carried out in the present work to match animal cells. These modifications included: 1- Using other laser system that used before, Excimer laser with 193 and 308 nm wavelengths. The used laser here, is He-Cd with low power and 441.5 nm wavelength in the visible region. 2- Instead of using pulsed laser, we used here CW He-Cd chopped by electrical chopper, which is synchronized with the mechanical motion of the mobile stage with step 40 microns, according to cell dimensions to avoid puncturing the same cell twice. The advantages of the modified here laser setup for gene transfer is: it is less damaging to the sensitive animal cell which has thin cell membrane. The present work aimed to: 1- Design a modified laser microbeam cell surgery, applicable to animal cells, such as fibroblast cells 2- To examine the efficiency of such system. 3- To assure gene transfer and its expression in the used cells. 4- To evaluate the ultra damages produced from using the laser beam as a modality for gene transfer. On the other wards, to introduce: safe, efficient and less damaging modality for gene transfer in animal cells. To achieve these goals, we applied the introduced here home-made laser setup with its synchronized parameters to introduce pBK-CMV phagemid, containing LacZ and neomycin resistance (neor )genes into BHK-21 fibroblast cell line. The results of the present work showed that: 1- Our modified laser microbeam cell surgery setup proved to be useful and efficient tool for gene transfer into fibroblast cells. 2- The presence and expression of LacZ gene was achieved using histochemical LacZ assay. 3- Selection of G418 antibiotic sensitivity assay confirmed the presence and expression towards stability of neor gene with time. 4- Presence of LacZ and neor genes in the genomic DNA of transfected fibroblast cells was indicated using PCR analysis. 5- Transmission electron microscopy indicated that, no ultradamages or changes for cell; membrane, organilles or any component of transfected fibroblast cell as a result of using laser microbeam compared with control cell.

Functional Characterization of a Strong Bi-directional Constitutive Plant Promoter Isolated from Cotton Leaf Curl Burewala Virus

PubMed Central

Khan, Zainul A.; Abdin, Malik Z.; Khan, Jawaid A.

2015-01-01

Cotton leaf curl Burewala virus (CLCuBuV), belonging to the genus Begomovirus, possesses single-stranded monopartite DNA genome. The bidirectional promoters representing Rep and coat protein (CP) genes of CLCuBuV were characterized and their efficacy was assayed. Rep and CP promoters of CLCuBuV and 35S promoter of Cauliflower mosaic virus (CaMV) were fused with β-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes. GUS activity in individual plant cells driven by Rep, CP and 35S promoters was estimated using real-time PCR and fluorometric GUS assay. Histochemical staining of GUS in transformed tobacco (Nicotiana tabacum cv. Xanthi) leaves showed highest expression driven by Rep promoter followed by 35S promoter and CP promoter. The expression level of GUS driven by Rep promoter in transformed tobacco plants was shown to be two to four-fold higher than that of 35S promoter, while the expression by CP promoter was slightly lower. Further, the expression of GFP was monitored in agroinfiltrated leaves of N. benthamiana, N. tabacum and cotton (Gossypium hirsutum) plants using confocal laser scanning microscopy. Rep promoter showed strong consistent transient expression in tobacco and cotton leaves as compared to 35S promoter. The strong constitutive CLCuBuV Rep promoter developed in this study could be very useful for high level expression of transgenes in a wide variety of plant cells. PMID:25799504

Functional characterization of a strong bi-directional constitutive plant promoter isolated from cotton leaf curl Burewala virus.

PubMed

Khan, Zainul A; Abdin, Malik Z; Khan, Jawaid A

2015-01-01

Cotton leaf curl Burewala virus (CLCuBuV), belonging to the genus Begomovirus, possesses single-stranded monopartite DNA genome. The bidirectional promoters representing Rep and coat protein (CP) genes of CLCuBuV were characterized and their efficacy was assayed. Rep and CP promoters of CLCuBuV and 35S promoter of Cauliflower mosaic virus (CaMV) were fused with β-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes. GUS activity in individual plant cells driven by Rep, CP and 35S promoters was estimated using real-time PCR and fluorometric GUS assay. Histochemical staining of GUS in transformed tobacco (Nicotiana tabacum cv. Xanthi) leaves showed highest expression driven by Rep promoter followed by 35S promoter and CP promoter. The expression level of GUS driven by Rep promoter in transformed tobacco plants was shown to be two to four-fold higher than that of 35S promoter, while the expression by CP promoter was slightly lower. Further, the expression of GFP was monitored in agroinfiltrated leaves of N. benthamiana, N. tabacum and cotton (Gossypium hirsutum) plants using confocal laser scanning microscopy. Rep promoter showed strong consistent transient expression in tobacco and cotton leaves as compared to 35S promoter. The strong constitutive CLCuBuV Rep promoter developed in this study could be very useful for high level expression of transgenes in a wide variety of plant cells.

Simultaneous visualization of the subfemtomolar expression of microRNA and microRNA target gene using HILO microscopy† †Electronic supplementary information (ESI) available: The LED device for the sample photobleaching, a schematic presentation of HILO microscopy, fluorescence spectra and hybridization curves of the molecular beacons, the linear correlation between the miRNA fluorescence intensity and the miRNA copy number, a validation of the miRNA adsorption and miRNA target gene expression via RT-qPCR, a validation of RT-qPCR using capillary electrophoresis, the reproducibility of RT-qPCR and Poisson distribution of the miRNA pipetting as well as a complete list of the oligonucleotides used in this study. See DOI: 10.1039/c7sc02701j Click here for additional data file.

PubMed Central

Lin, Yi-Zhen; Ou, Da-Liang; Chang, Hsin-Yuan; Lin, Wei-Yu; Hsu, Chiun

2017-01-01

The family of microRNAs (miRNAs) not only plays an important role in gene regulation but is also useful for the diagnosis of diseases. A reliable method with high sensitivity may allow researchers to detect slight fluctuations in ultra-trace amounts of miRNA. In this study, we propose a sensitive imaging method for the direct probing of miR-10b (miR-10b-3p, also called miR-10b*) and its target (HOXD10 mRNA) in fixed cells based on the specific recognition of molecular beacons combined with highly inclined and laminated optical sheet (HILO) fluorescence microscopy. The designed dye-quencher-labelled molecular beacons offer excellent efficiencies of fluorescence resonance energy transfer that allow us to detect miRNA and the target mRNA simultaneously in hepatocellular carcinoma cells using HILO fluorescence microscopy. Not only can the basal trace amount of miRNA be observed in each individual cell, but the obtained images also indicate that this method is useful for monitoring the fluctuations in ultra-trace amounts of miRNA when the cells are transfected with a miRNA precursor or a miRNA inhibitor (anti-miR). Furthermore, a reasonable causal relation between the miR-10b and HOXD10 expression levels was observed in miR-10b* precursor-transfected cells and miR-10b* inhibitor-transfected cells. The trends of the miRNA alterations obtained using HILO microscopy completely matched the RT-qPCR data and showed remarkable reproducibility (the coefficient of variation [CV] = 0.86%) and sensitivity (<1.0 fM). This proposed imaging method appears to be useful for the simultaneous visualisation of ultra-trace amounts of miRNA and target mRNA and excludes the procedures for RNA extraction and amplification. Therefore, the visualisation of miRNA and the target mRNA should facilitate the exploration of the functions of ultra-trace amounts of miRNA in fixed cells in biological studies and may serve as a powerful tool for diagnoses based on circulating cancer cells. PMID:28989695

The Pochonia chlamydosporia serine protease gene vcp1 is subject to regulation by carbon, nitrogen and pH: implications for nematode biocontrol.

PubMed

Ward, Elaine; Kerry, Brian R; Manzanilla-López, Rosa H; Mutua, Gerald; Devonshire, Jean; Kimenju, John; Hirsch, Penny R

2012-01-01

The alkaline serine protease VCP1 of the fungus Pochonia chlamydosporia belongs to a family of subtilisin-like enzymes that are involved in infection of nematode and insect hosts. It is involved early in the infection process, removing the outer proteinaceous vitelline membrane of nematode eggs. Little is known about the regulation of this gene, even though an understanding of how nutrients and other factors affect its expression is critical for ensuring its efficacy as a biocontrol agent. This paper provides new information on the regulation of vcp1 expression. Sequence analysis of the upstream regulatory region of this gene in 30 isolates revealed that it was highly conserved and contained sequence motifs characteristic of genes that are subject to carbon, nitrogen and pH-regulation. Expression studies, monitoring enzyme activity and mRNA, confirmed that these factors affect VCP1 production. As expected, glucose reduced VCP1 expression and for a few hours so did ammonium chloride. Surprisingly, however, by 24 h VCP1 levels were increased in the presence of ammonium chloride for most isolates. Ambient pH also regulated VCP1 expression, with most isolates producing more VCP1 under alkaline conditions. There were some differences in the response of one isolate with a distinctive upstream sequence including a variant regulatory-motif profile. Cryo-scanning electron microscopy studies indicated that the presence of nematode eggs stimulates VCP1 production by P. chlamydosporia, but only where the two are in close contact. Overall, the results indicate that readily-metabolisable carbon sources and unfavourable pH in the rhizosphere/egg-mass environment may compromise nematode parasitism by P. chlamydosporia. However, contrary to previous indications using other nematophagous and entomopathogenic fungi, ammonium nitrate (e.g. from fertilizers) may enhance biocontrol potential in some circumstances.

Binding and condensation of plasmid DNA onto functionalized carbon nanotubes: toward the construction of nanotube-based gene delivery vectors.

PubMed

Singh, Ravi; Pantarotto, Davide; McCarthy, David; Chaloin, Olivier; Hoebeke, Johan; Partidos, Charalambos D; Briand, Jean-Paul; Prato, Maurizio; Bianco, Alberto; Kostarelos, Kostas

2005-03-30

Carbon nanotubes (CNTs) constitute a class of nanomaterials that possess characteristics suitable for a variety of possible applications. Their compatibility with aqueous environments has been made possible by the chemical functionalization of their surface, allowing for exploration of their interactions with biological components including mammalian cells. Functionalized CNTs (f-CNTs) are being intensively explored in advanced biotechnological applications ranging from molecular biosensors to cellular growth substrates. We have been exploring the potential of f-CNTs as delivery vehicles of biologically active molecules in view of possible biomedical applications, including vaccination and gene delivery. Recently we reported the capability of ammonium-functionalized single-walled CNTs to penetrate human and murine cells and facilitate the delivery of plasmid DNA leading to expression of marker genes. To optimize f-CNTs as gene delivery vehicles, it is essential to characterize their interactions with DNA. In the present report, we study the interactions of three types of f-CNTs, ammonium-functionalized single-walled and multiwalled carbon nanotubes (SWNT-NH3+; MWNT-NH3+), and lysine-functionalized single-walled carbon nanotubes (SWNT-Lys-NH3+), with plasmid DNA. Nanotube-DNA complexes were analyzed by scanning electron microscopy, surface plasmon resonance, PicoGreen dye exclusion, and agarose gel shift assay. The results indicate that all three types of cationic carbon nanotubes are able to condense DNA to varying degrees, indicating that both nanotube surface area and charge density are critical parameters that determine the interaction and electrostatic complex formation between f-CNTs with DNA. All three different f-CNT types in this study exhibited upregulation of marker gene expression over naked DNA using a mammalian (human) cell line. Differences in the levels of gene expression were correlated with the structural and biophysical data obtained for the f-CNT:DNA complexes to suggest that large surface area leading to very efficient DNA condensation is not necessary for effective gene transfer. However, it will require further investigation to determine whether the degree of binding and tight association between DNA and nanotubes is a desirable trait to increase gene expression efficiency in vitro or in vivo. This study constitutes the first thorough investigation into the physicochemical interactions between cationic functionalized carbon nanotubes and DNA toward construction of carbon nanotube-based gene transfer vector systems.

Long term storage in liquid nitrogen leads to only minor phenotypic and gene expression changes in the mammary carcinoma model cell line BT474.

PubMed

Fazekas, Judit; Grunt, Thomas W; Jensen-Jarolim, Erika; Singer, Josef

2017-05-23

Cancer cell lines are indispensible surrogate models in cancer research, as they can be used off-the-shelf, expanded to the desired extent, easily modified and exchanged between research groups for affirmation, reproduction or follow-up experiments.As malignant cells are prone to genomic instability, phenotypical changes may occur after certain passages in culture. Thus, cell lines have to be regularly authenticated to ensure data quality. In between experiments these cell lines are often stored in liquid nitrogen for extended time periods.Although freezing of cells is a necessary evil, little research is performed on how long-term storage affects cancer cell lines. Therefore, this study investigated the effects of a 28-year long liquid nitrogen storage period on BT474 cells with regard to phenotypical changes, differences in cell-surface receptor expression as well as cytokine and gene expressional variations. Two batches of BT474 cells, one frozen in 1986, the other directly purchased from ATCC were investigated by light microscopy, cell growth analysis, flow cytometry and cytokine as well as whole-transcriptome expression profiling. The cell lines were morphologically indifferent and showed similar growth rates and similar cell-surface receptor expression. Transcriptome analysis revealed significant differences in only 26 of 40,716 investigated RefSeq transcripts with 4 of them being up-regulated and 22 down-regulated. This study demonstrates that even after very long periods of storage in liquid nitrogen, cancer cell lines display only minimal changes in their gene expression profiles. However, also such minor changes should be carefully assessed before continuation of experiments, especially if phenotypic alterations can be additionally observed.

Pathogenic Leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily

PubMed Central

Matsunaga, James; Barocchi, Michele A.; Croda, Julio; Young, Tracy A.; Sanchez, Yolanda; Siqueira, Isadora; Bolin, Carole A.; Reis, Mitermayer G.; Riley, Lee W.; Haake, David A.; Ko, Albert I.

2005-01-01

Summary Proteins with bacterial immunoglobulin-like (Big) domains, such as the Yersinia pseudotuberculosis invasin and Escherichia coli intimin, are surface-expressed proteins that mediate host mammalian cell invasion or attachment. Here, we report the identification and characterization of a new family of Big domain proteins, referred to as Lig (leptospiral Ig-like) proteins, in pathogenic Leptospira. Screening of L. interrogans and L. kirschneri expression libraries with sera from leptospirosis patients identified 13 lambda phage clones that encode tandem repeats of the 90 amino acid Big domain. Two lig genes, designated ligA and ligB, and one pseudo-gene, ligC, were identified. The ligA and ligB genes encode amino-terminal lipoprotein signal peptides followed by 10 or 11 Big domain repeats and, in the case of ligB, a unique carboxy-terminal non-repeat domain. The organization of ligC is similar to that of ligB but contains mutations that disrupt the reading frame. The lig sequences are present in pathogenic but not saprophytic Leptospira species. LigA and LigB are expressed by a variety of virulent leptospiral strains. Loss of Lig protein and RNA transcript expression is correlated with the observed loss of virulence during culture attenuation of pathogenic strains. High-pressure freeze substitution followed by immunocytochemical electron microscopy confirmed that the Lig proteins were localized to the bacterial surface. Immunoblot studies with patient sera found that the Lig proteins are a major antigen recognized during the acute host infection. These observations demonstrate that the Lig proteins are a newly identified surface protein of pathogenic Leptospira, which by analogy to other bacterial immunoglobulin superfamily virulence factors, may play a role in host cell attachment and invasion during leptospiral pathogenesis. PMID:12890019

A non-BRICHOS SFTPC mutant (SP-CI73T) linked to interstitial lung disease promotes a late block in macroautophagy disrupting cellular proteostasis and mitophagy

PubMed Central

Hawkins, Arie; Guttentag, Susan H.; Deterding, Robin; Funkhouser, William K.; Goralski, Jennifer L.; Chatterjee, Shampa; Beers, Michael F.

2014-01-01

Mutation of threonine for isoleucine at codon 73 (I73T) in the human surfactant protein C (hSP-C) gene (SFTPC) accounts for a significant portion of SFTPC mutations associated with interstitial lung disease (ILD). Cell lines stably expressing tagged primary translation product of SP-C isoforms were generated to test the hypothesis that deposition of hSP-CI73T within the endosomal system promotes disruption of a key cellular quality control pathway, macroautophagy. By fluorescence microscopy, wild-type hSP-C (hSP-CWT) colocalized with exogenously expressed human ATP binding cassette class A3 (hABCA3), an indicator of normal trafficking to lysosomal-related organelles. In contrast, hSP-CI73T was dissociated from hABCA3 but colocalized to the plasma membrane as well as the endosomal network. Cells expressing hSP-CI73T exhibited increases in size and number of cytosolic green fluorescent protein/microtubule-associated protein 1 light-chain 3 (LC3) vesicles, some of which colabeled with red fluorescent protein from the gene dsRed/hSP-CI73T. By transmission electron microscopy, hSP-CI73T cells contained abnormally large autophagic vacuoles containing organellar and proteinaceous debris, which phenocopied ultrastructural changes in alveolar type 2 cells in a lung biopsy from a SFTPC I73T patient. Biochemically, hSP-CI73T cells exhibited increased expression of Atg8/LC3, SQSTM1/p62, and Rab7, consistent with a distal block in autophagic vacuole maturation, confirmed by flux studies using bafilomycin A1 and rapamycin. Functionally, hSP-CI73T cells showed an impaired degradative capacity for an aggregation-prone huntingtin-1 reporter substrate. The disruption of autophagy-dependent proteostasis was accompanied by increases in mitochondria biomass and parkin expression coupled with a decrease in mitochondrial membrane potential. We conclude that hSP-CI73T induces an acquired block in macroautophagy-dependent proteostasis and mitophagy, which could contribute to the increased vulnerability of the lung epithelia to second-hit injury as seen in ILD. PMID:25344067

Structural and functional assessment of intense therapeutic ultrasound effects on partial Achilles tendon transection

NASA Astrophysics Data System (ADS)

Barton, Jennifer K.; Rice, Photini S.; Howard, Caitlin C.; Koevary, Jen W.; Danford, Forest; Gonzales, David A.; Vande Geest, Jon; Latt, L. Daniel; Szivek, John A.; Amodei, Richard; Slayton, Michael

2018-02-01

Tendinopathies and tendon tears heal slowly because tendons have a limited blood supply. Intense therapeutic ultrasound (ITU) is a treatment modality that creates very small, focal coagula in tissue, which can stimulate a healing response. This pilot study investigated the effects of ITU on rabbit and rat models of partial Achilles tendon rupture. The right Achilles tendons of 20 New Zealand White rabbits and 118 rats were partially transected. Twenty-four hours after surgery, ITU coagula were placed in the tendon and surrounding tissue, alternating right and left legs. At various time points, the following data were collected: ultrasound imaging, optical coherence tomography (OCT) imaging, mechanical testing, gene expression analysis, histology, and multiphoton microscopy (MPM) of sectioned tissue. Ultrasound visualized cuts and treatment lesions. OCT showed the effect of the interventions on birefringence banding caused by collagen organization. MPM showed inflammatory infiltrate, collagen synthesis and organization. By day 14- 28, all tendons had a smooth appearance and histology, MPM and OCT still could still visualize residual healing processes. Few significant results in gene expression were seen, but trends were that ITU treatment caused an initial decrease in growth and collagen gene expression followed by an increase. No difference in failure loads was found between control, cut, and ITU treatment groups, suggesting that sufficient healing had occurred by 14 days to restore all test tissue to control mechanical properties. These results suggest that ITU does not cause harm to tendon tissue. Upregulation of some genes suggests that ITU may increase healing response.

A Critical Role of Mitochondria in BDNF-Associated Synaptic Plasticity After One-Week Vortioxetine Treatment.

PubMed

Chen, Fenghua; Danladi, Jibrin; Ardalan, Maryam; Elfving, Betina; Müller, Heidi K; Wegener, Gregers; Sanchez, Connie; Nyengaard, Jens R

2018-06-01

Preclinical studies have indicated that antidepressant effect of vortioxetine involves increased synaptic plasticity and promotion of spine maturation. Mitochondria dysfunction may contribute to the pathophysiological basis of major depressive disorder. Taking into consideration that vortioxetine increases spine number and dendritic branching in hippocampus CA1 faster than fluoxetine, we hypothesize that new spines induced by vortioxetine can rapidly form functional synapses by mitochondrial support, accompanied by increased brain-derived neurotrophic factor signaling. Rats were treated for 1 week with vortioxetine or fluoxetine at pharmacologically relevant doses. Number of synapses and mitochondria in hippocampus CA1 were quantified by electron microscopy. Brain-derived neurotrophic factor protein levels were visualized with immunohistochemistry. Gene and protein expression of synapse and mitochondria-related markers were investigated with real-time quantitative polymerase chain reaction and immunoblotting. Vortioxetine increased number of synapses and mitochondria significantly, whereas fluoxetine had no effect after 1-week dosing. BDNF levels in hippocampus DG and CA1 were significantly higher after vortioxetine treatment. Gene expression levels of Rac1 after vortioxetine treatment were significantly increased. There was a tendency towards increased gene expression levels of Drp1 and protein levels of Rac1. However, both gene and protein levels of c-Fos were significantly decreased. Furthermore, there was a significant positive correlation between BDNF levels and mitochondria and synapse numbers. Our results imply that mitochondria play a critical role in synaptic plasticity accompanied by increased BDNF levels. Rapid changes in BDNF levels and synaptic/mitochondria plasticity of hippocampus following vortioxetine compared with fluoxetine may be ascribed to vortioxetine's modulation of serotonin receptors.

A Fluorescent Bioreporter for Acetophenone and 1-Phenylethanol derived from a Specifically Induced Catabolic Operon.

PubMed

Muhr, Enrico; Leicht, Oliver; González Sierra, Silvia; Thanbichler, Martin; Heider, Johann

2015-01-01

The β-proteobacterium Aromatoleum aromaticum degrades the aromatic ketone acetophenone, a key intermediate of anaerobic ethylbenzene metabolism, either aerobically or anaerobically via a complex ATP-dependent acetophenone carboxylase and a benzoylacetate-CoA ligase. The genes coding for these enzymes (apcABCDE and bal) are organized in an apparent operon and are expressed in the presence of the substrate acetophenone. To study the conditions under which this operon is expressed in more detail, we constructed a reporter strain by inserting a gene fusion of apcA, the first gene of the apc-bal operon, with the gene for the fluorescent protein mCherry into the chromosome of A. aromaticum. The fusion protein indeed accumulated consistently with the expression pattern of the acetophenone-metabolic enzymes under various growth conditions. After evaluating and quantifying the data by fluorescence microscopy, fluorescence-based flow cytometry and immunoblot analysis, mCherry production was found to be proportional to the applied acetophenone concentrations. The reporter strain allowed quantification of acetophenone within a concentration range of 50 μM (detection limit) to 250 μM after 12 and 24 h. Moreover, production of the Apc-mCherry fusion protein in the reporter strain was highly specific and responded to acetophenone and both enantiomers of 1-phenylethanol, which are easily converted to acetophenone. Other analogous substrates showed either a significantly weaker response or none at all. Therefore, the reporter strain provides a basis for the development of a specific bioreporter system for acetophenone with an application potential reaching from environmental monitoring to petroleum prospecting.

A Fluorescent Bioreporter for Acetophenone and 1-Phenylethanol derived from a Specifically Induced Catabolic Operon

PubMed Central

Muhr, Enrico; Leicht, Oliver; González Sierra, Silvia; Thanbichler, Martin; Heider, Johann

2016-01-01

The β-proteobacterium Aromatoleum aromaticum degrades the aromatic ketone acetophenone, a key intermediate of anaerobic ethylbenzene metabolism, either aerobically or anaerobically via a complex ATP-dependent acetophenone carboxylase and a benzoylacetate-CoA ligase. The genes coding for these enzymes (apcABCDE and bal) are organized in an apparent operon and are expressed in the presence of the substrate acetophenone. To study the conditions under which this operon is expressed in more detail, we constructed a reporter strain by inserting a gene fusion of apcA, the first gene of the apc-bal operon, with the gene for the fluorescent protein mCherry into the chromosome of A. aromaticum. The fusion protein indeed accumulated consistently with the expression pattern of the acetophenone-metabolic enzymes under various growth conditions. After evaluating and quantifying the data by fluorescence microscopy, fluorescence-based flow cytometry and immunoblot analysis, mCherry production was found to be proportional to the applied acetophenone concentrations. The reporter strain allowed quantification of acetophenone within a concentration range of 50 μM (detection limit) to 250 μM after 12 and 24 h. Moreover, production of the Apc-mCherry fusion protein in the reporter strain was highly specific and responded to acetophenone and both enantiomers of 1-phenylethanol, which are easily converted to acetophenone. Other analogous substrates showed either a significantly weaker response or none at all. Therefore, the reporter strain provides a basis for the development of a specific bioreporter system for acetophenone with an application potential reaching from environmental monitoring to petroleum prospecting. PMID:26858693

Unprecedented Silver Resistance in Clinically Isolated Enterobacteriaceae: Major Implications for Burn and Wound Management

PubMed Central

Norton, Rhy; Austin, Cindy; Mitchell, Amber; Zank, Sara; Durham, Paul

2015-01-01

Increased utilization of inorganic silver as an adjunctive to many medical devices has raised concerns of emergent silver resistance in clinical bacteria. Although the molecular basis for silver resistance has been previously characterized, to date, significant phenotypic expression of these genes in clinical settings is yet to be observed. Here, we identified the first strains of clinical bacteria expressing silver resistance at a level that could significantly impact wound care and the use of silver-based dressings. Screening of 859 clinical isolates confirmed 31 harbored at least 1 silver resistance gene. Despite the presence of these genes, MIC testing revealed most of the bacteria displayed little or no increase in resistance to ionic silver (200 to 300 μM Ag+). However, 2 isolates (Klebsiella pneumonia and Enterobacter cloacae) were capable of robust growth at exceedingly high silver concentrations, with MIC values reaching 5,500 μM Ag+. DNA sequencing of these two strains revealed the presence of genes homologous to known genetic determinants of heavy metal resistance. Darkening of the bacteria's pigment was observed after exposure to high silver concentrations. Scanning electron microscopy images showed the presence of silver nanoparticles embedded in the extracellular polymeric substance of both isolates. This finding suggested that the isolates may neutralize ionic silver via reduction to elemental silver. Antimicrobial testing revealed both organisms to be completely resistant to many commercially available silver-impregnated burn and wound dressings. Taken together, these findings provide the first evidence of clinical bacteria capable of expressing silver resistance at levels that could significantly impact wound management. PMID:26014954

Oxidized ultrashort nanotubes as carbon scaffolds for the construction of cell-penetrating NF-kappaB decoy molecules.

PubMed

Crinelli, Rita; Carloni, Elisa; Menotta, Michele; Giacomini, Elisa; Bianchi, Marzia; Ambrosi, Gianluca; Giorgi, Luca; Magnani, Mauro

2010-05-25

Oligonucleotide (ODN) decoys are synthetic ODNs containing the DNA binding sequence of a transcription factor. When delivered to cells, these molecules can compete with endogenous sequences for binding the transcription factor, thus inhibiting its ability to activate the expression of target genes. Modulation of gene expression by decoy ODNs against nuclear factor-kappaB (NF-kappaB), a transcription factor regulating many genes involved in immunity, has been achieved in a variety of immune/inflammatory disorders. However, the successful use of transcription factor decoys depends on an efficient means to bring the synthetic DNA to target cells. It is known that single-walled carbon nanotubes (SWCNTs), under certain conditions, are able to cross the cell membrane. Thus, we have evaluated the possibility to functionalize SWCNTs with decoy ODNs against NF-kappaB in order to improve their intracellular delivery. To couple ODNs to CNTs, we have exploited the carbodiimide chemistry which allows covalent binding of amino-modified ODNs to carboxyl groups introduced onto SWCNTs through oxidation. The effective binding of ODNs to nanotubes has been demonstrated by a combination of microscopic, spectroscopic, and electrophoretic techniques. The uptake and subcellular distribution of ODN decoys bound to SWCNTs was analyzed by fluorescence microscopy. ODNs were internalized into macrophages and accumulated in the cytosol. Moreover, no cytotoxicity associated with SWCNT administration was observed. Finally, NF-kappaB-dependent gene expression was significantly reduced in cells receiving nanomolar concentrations of SWCNT-NF-kappaB decoys compared to cells receiving SWCNTs or SWCNTs functionalized with a nonspecific ODN sequence, demonstrating both efficacy and specificity of the approach.

Integration of information and volume visualization for analysis of cell lineage and gene expression during embryogenesis

NASA Astrophysics Data System (ADS)

Cedilnik, Andrej; Baumes, Jeffrey; Ibanez, Luis; Megason, Sean; Wylie, Brian

2008-01-01

Dramatic technological advances in the field of genomics have made it possible to sequence the complete genomes of many different organisms. With this overwhelming amount of data at hand, biologists are now confronted with the challenge of understanding the function of the many different elements of the genome. One of the best places to start gaining insight on the mechanisms by which the genome controls an organism is the study of embryogenesis. There are multiple and inter-related layers of information that must be established in order to understand how the genome controls the formation of an organism. One is cell lineage which describes how patterns of cell division give rise to different parts of an organism. Another is gene expression which describes when and where different genes are turned on. Both of these data types can now be acquired using fluorescent laser-scanning (confocal or 2-photon) microscopy of embryos tagged with fluorescent proteins to generate 3D movies of developing embryos. However, analyzing the wealth of resulting images requires tools capable of interactively visualizing several different types of information as well as being scalable to terabytes of data. This paper describes how the combination of existing large data volume visualization and the new Titan information visualization framework of the Visualization Toolkit (VTK) can be applied to the problem of studying the cell lineage of an organism. In particular, by linking the visualization of spatial and temporal gene expression data with novel ways of visualizing cell lineage data, users can study how the genome regulates different aspects of embryonic development.

[5-aza-2'-deoxycytidine-induced inhibition of CDH13 expression and its inhibitory effect on methylation status in human colon cancer cells in vitro and on growth of xenograft in nude mice].

PubMed

Ren, Jian-zhen; Huo, Ji-rong

2012-01-01

To determine the inhibitory effect of 5-aza-2'-deoxycytidine (5-Aza-CdR) on the growth of human colon carcinoma cells and xenografts in nude mice, to observe its effect on CDH13 gene expression and methylation in the xenografts, and to explore the possible mechanisms. Human colon carcinoma cell line HCT116 cells were treated with 5-Aza-CdR, and the cell morphology was observe by phase contrast microscopy. The cell growth was assessed by MTT assay. A tumor-bearing mouse model was generated by subcutaneous inoculation of human colon carcinoma HCT116 cells into nude mice. The tumor growth in the nude mice was observed, the CDH13 gene expression and its methylation status in the tumors were detected using methylation specific PCR (MSP), RT-PCR, Western blotting and immunohistochemistry. After treatment with 5-Aza-CdR, the inhibition rate of the growth of cultured HCT116 cells was increased as the concentration was increasing. The growth of the xenografts in nude mice was significantly inhibited, and the methylated CDH13 gene was reactivated. After 4 weeks of 5-Aza-CdR treatment, no significant difference was found between the body weights of nude mice in the 5-Aza-CdR group [(18.06 ± 1.29) g] and control group [(17.07 ± 0.84) g], (P > 0.10), and the average volume of xenografts of the 5-Aza-CdR group was (907.00 ± 87.29) mm(3), significantly smaller than the (1370.93 ± 130.20) mm(3) in the control group (P < 0.005). No expression of CDH13 gene was found in the control group. The expression of CDH13 gene in the 5-Aza-CdR group was increased along with the increasing concentration of 5-Aza-CdR. 5-Aza-CdR inhibits the growth of human colon cancer cells in culture and in nude mice, and induces the cancer cells to re-express CDH13 in nude mice. Its mechanism may be that demethylation of the methylated CDH13 promoter induced by 5-Aza-CdR restores CDH13 expression and thus inhibits the tumor growth in nude mice.

Mechanisms and dynamics of nuclear lamina-genome interactions.

PubMed

Amendola, Mario; van Steensel, Bas

2014-06-01

The nuclear lamina (NL) interacts with the genomic DNA and is thought to influence chromosome organization and gene expression. Both DNA sequences and histone modifications are important for NL tethering of the genomic DNA. These interactions are dynamic in individual cells and can change during differentiation and development. Evidence is accumulating that the NL contributes to the repression of transcription. Advances in mapping, genome-editing and microscopy techniques are increasing our understanding of the molecular mechanisms involved in NL-genome interactions. Copyright © 2014 Elsevier Ltd. All rights reserved.

Increased Expression of the Innate Immune Receptor TLR10 in Obesity and Type-2 Diabetes: Association with ROS-Mediated Oxidative Stress.

PubMed

Sindhu, Sardar; Akhter, Nadeem; Kochumon, Shihab; Thomas, Reeby; Wilson, Ajit; Shenouda, Steve; Tuomilehto, Jaakko; Ahmad, Rasheed

2018-01-01

Metabolic diseases such as obesity and type-2 diabetes (T2D) are known to be associated with chronic low-grade inflammation called metabolic inflammation together with an oxidative stress milieu found in the expanding adipose tissue. The innate immune Toll-like receptors (TLR) such as TLR2 and TLR4 have emerged as key players in metabolic inflammation; nonetheless, TLR10 expression in the adipose tissue and its significance in obesity/T2D remain unclear. TLR10 gene expression was determined in the adipose tissue samples from healthy non-diabetic and T2D individuals, 13 each, using real-time RT-PCR. TLR10 protein expression was determined by immunohistochemistry, confocal microscopy, and flow cytometry. Regarding in vitro studies, THP-1 cells, peripheral blood mononuclear cells (PBMC), or primary monocytes were treated with hydrogen peroxide (H2O2) for induction of reactive oxygen species (ROS)-mediated oxidative stress. Superoxide dismutase (SOD) activity was measured using a commercial kit. Data (mean±SEM) were compared using unpaired student's t-test and P<0.05 was considered significant. The adipose tissue TLR10 gene/protein expression was found to be significantly upregulated in obesity as well as T2D which correlated with body mass index (BMI). ROS-mediated oxidative stress induced high levels of TLR10 gene/protein expression in monocytic cells and PBMC. In these cells, oxidative stress induced a time-dependent increase in SOD activity. Pre-treatment of cells with anti-oxidants/ROS scavengers diminished the expression of TLR10. ROS-induced TLR10 expression involved the nuclear factor-kappaB (NF-κB)/mitogen activated protein kinase (MAPK) signaling as well as endoplasmic reticulum (ER) stress. H2O2-induced oxidative stress interacted synergistically with palmitate to trigger the expression of TLR10 which associated with enhanced expression of proinflammatory cytokines/chemokine. Oxidative stress induces the expression of TLR10 which may represent an immune marker for metabolic inflammation. © 2018 The Author(s). Published by S. Karger AG, Basel.

Polyethyleneimine-capped silver nanoclusters for microRNA oligonucleotide delivery and bacterial inhibition

PubMed Central

Liang, Jichao; Luo, Ailing; Wang, Lingqian; Zhu, Jing; Xiong, Huayu; Chen, Yong

2017-01-01

Efficient and safe nonviral gene delivery systems are a prerequisite for the clinical application of therapeutic genes. In this paper, polyethyleneimine-capped silver nanoclusters (PEI-AgNCs) were prepared for the purpose of microRNA (miRNA) delivery. The resultant PEI-AgNCs were characterized by a photoluminescence assay and transmission electron microscopy. A cytotoxicity assay showed that PEI-AgNCs exhibit relatively low cytotoxicity. Interestingly, PEI-AgNCs were confirmed to transfect miRNA mimics more effectively than PEI in HepG2 and 293A cells. In this regard, hsa-miR-21 or hsa-miR-221 mimics (miR-21/221m) were transported into HepG2 cells by using PEI-AgNCs. The miR-21/221 expression was determined post-transfection by quantitative real-time polymerase chain reaction. Compared with the negative control, PEI-AgNCs/miR-21/221m groups exhibited higher miR-21/221 levels. In addition, AgNCs endow PEI with stronger antibacterial activity, and this advantage provided PEI-AgNCs the potential to prevent bacterial contamination during the transfection process. Furthermore, we showed that PEI-AgNCs are viable nanomaterials for plain imaging of the cells by laser scanning confocal microscopy, indicating great potential as an ideal fluorescent probe to track the transfection behavior. These results demonstrated that PEI-AgNCs are promising and novel nonviral vectors for gene delivery. PMID:29238194

dsRNA silencing of an R2R3-MYB transcription factor affects flower cell shape in a Dendrobium hybrid.

PubMed

Lau, Su-Ee; Schwarzacher, Trude; Othman, Rofina Yasmin; Harikrishna, Jennifer Ann

2015-08-11

The R2R3-MYB genes regulate pigmentation and morphogenesis of flowers, including flower and cell shape, and therefore have importance in the development of new varieties of orchids. However, new variety development is limited by the long breeding time required in orchids. In this study, we identified a cDNA, DhMYB1, that is expressed during flower development in a hybrid orchid, Dendrobium hybrida (Dendrobium bobby messina X Dendrobium chao phraya) then used the direct application of dsRNA to observe the effect of gene silencing on flower phenotype and floral epidermal cell shape. Flower bud development in the Dendrobium hybrid was characterised into seven stages and the time of meiosis was determined as between stages 3 to 5 when the bud is approximately half of the mature size. Scanning electron microscopy characterisation of adaxial epidermal cells of the flower perianth, showed that the petals and sepals each are divided into two distinct domains based on cell shape and size, while the labellum comprises seven domains. Thirty-two partial cDNA fragments representing R2R3-MYB gene sequences were isolated from D. hybrida. Phylogenetic analysis revealed that nine of the translated sequences were clustered with MYB sequences that are known to be involved in cell shape development and from these, DhMYB1 was selected for full length cDNA cloning and functional study. Direct application of a 430 bp dsRNA from the 3' region of DhMYB1 to emerging orchid flower buds reduced expression of DhMYB1 RNA compared with untreated control. Scanning electron microscopy of adaxial epidermal cells within domain one of the labellum of flowers treated with DhMYB1 dsRNA showed flattened epidermal cells whilst those of control flowers were conical. DhMYB1 is expressed throughout flower bud development and is involved in the development of the conical cell shape of the epidermal cells of the Dendrobium hybrida flower labellum. The direct application of dsRNA changed the phenotype of floral cells, thus, this technique may have application in floriculture biotechnology.

Inflammatory cascades mediate synapse elimination in spinal cord compression

PubMed Central

2014-01-01

Background Cervical compressive myelopathy (CCM) is caused by chronic spinal cord compression due to spondylosis, a degenerative disc disease, and ossification of the ligaments. Tip-toe walking Yoshimura (twy) mice are reported to be an ideal animal model for CCM-related neuronal dysfunction, because they develop spontaneous spinal cord compression without any artificial manipulation. Previous histological studies showed that neurons are lost due to apoptosis in CCM, but the mechanism underlying this neurodegeneration was not fully elucidated. The purpose of this study was to investigate the pathophysiology of CCM by evaluating the global gene expression of the compressed spinal cord and comparing the transcriptome analysis with the physical and histological findings in twy mice. Methods Twenty-week-old twy mice were divided into two groups according to the magnetic resonance imaging (MRI) findings: a severe compression (S) group and a mild compression (M) group. The transcriptome was analyzed by microarray and RT-PCR. The cellular pathophysiology was examined by immunohistological analysis and immuno-electron microscopy. Motor function was assessed by Rotarod treadmill latency and stride-length tests. Results Severe cervical calcification caused spinal canal stenosis and low functional capacity in twy mice. The microarray analysis revealed 215 genes that showed significantly different expression levels between the S and the M groups. Pathway analysis revealed that genes expressed at higher levels in the S group were enriched for terms related to the regulation of inflammation in the compressed spinal cord. M1 macrophage-dominant inflammation was present in the S group, and cysteine-rich protein 61 (Cyr61), an inducer of M1 macrophages, was markedly upregulated in these spinal cords. Furthermore, C1q, which initiates the classical complement cascade, was more upregulated in the S group than in the M group. The confocal and electron microscopy observations indicated that classically activated microglia/macrophages had migrated to the compressed spinal cord and eliminated synaptic terminals. Conclusions We revealed the detailed pathophysiology of the inflammatory response in an animal model of chronic spinal cord compression. Our findings suggest that complement-mediated synapse elimination is a central mechanism underlying the neurodegeneration in CCM. PMID:24589419

Study of stem cell homing & self-renewal marker gene profile of ex vivo expanded human CD34+ cells manipulated with a mixture of cytokines & stromal cell-derived factor 1

PubMed Central

Kode, Jyoti; Khattry, Navin; Bakshi, Ashish; Amrutkar, Vasanti; Bagal, Bhausaheb; Karandikar, Rohini; Rane, Pallavi; Fujii, Nobutaka; Chiplunkar, Shubhada

2017-01-01

Background & objectives: Next generation transplantation medicine aims to develop stimulating cocktail for increased ex vivo expansion of primitive hematopoietic stem and progenitor cells (HSPC). The present study was done to evaluate the cocktail GF (Thrombopoietin + Stem Cell factor + Flt3-ligand) and homing-defining molecule Stromal cell-derived factor 1 (SDF1) for HSPC ex vivo expansion. Methods: Peripheral blood stem cell (n=74) harvests were analysed for CD34hi CD45lo HSPC. Immunomagnetically enriched HSPC were cultured for eight days and assessed for increase in HSPC, colony forming potential in vitro and in vivo engrafting potential by analyzing human CD45+ cells. Expression profile of genes for homing and stemness were studied using microarray analysis. Expression of adhesion/homing markers were validated by flow cytometry/ confocal microscopy. Results: CD34hi CD45lo HSPC expansion cultures with GF+SDF1 demonstrated increased nucleated cells (n=28, P< 0.001), absolute CD34+ cells (n=8, P=0.021) and increased colony forming units (cfu) compared to unstimulated and GF-stimulated HSPC. NOD-SCID mice transplanted with GF+SDF1-HSPC exhibited successful homing/engraftment (n=24, P< 0.001). Microarray analysis of expanded HSPC demonstrated increased telomerase activity and many homing-associated genes (35/49) and transcription factors for stemness/self-renewal (49/56) were significantly upregulated in GF+SDF1 stimulated HSPC when compared to GF-stimulated HSPC. Expression of CD44, CXCR4, CD26, CD14, CD45 and soluble IL-6 in expanded cultures were validated by flow cytometry and confocal microscopy. Interpretation & conclusions: Cocktail of cytokines and SDF1 showed good potential to successfully expand HSPC which exhibited enhanced ability to generate multilineage cells in short-term and long-term repopulation assay. This cocktail-mediated stem cell expansion has potential to obviate the need for longer and large volume apheresis procedure making it convenient for donors. PMID:29168461

Amastin Knockdown in Leishmania braziliensis Affects Parasite-Macrophage Interaction and Results in Impaired Viability of Intracellular Amastigotes.

PubMed

de Paiva, Rita Marcia Cardoso; Grazielle-Silva, Viviane; Cardoso, Mariana Santos; Nakagaki, Brenda Naemi; Mendonça-Neto, Rondon Pessoa; Canavaci, Adriana Monte Cassiano; Souza Melo, Normanda; Martinelli, Patrícia Massara; Fernandes, Ana Paula; daRocha, Wanderson Duarte; Teixeira, Santuza M R

2015-12-01

Leishmaniasis, a human parasitic disease with manifestations ranging from cutaneous ulcerations to fatal visceral infection, is caused by several Leishmania species. These protozoan parasites replicate as extracellular, flagellated promastigotes in the gut of a sandfly vector and as amastigotes inside the parasitophorous vacuole of vertebrate host macrophages. Amastins are surface glycoproteins encoded by large gene families present in the genomes of several trypanosomatids and highly expressed in the intracellular amastigote stages of Trypanosoma cruzi and Leishmania spp. Here, we showed that the genome of L. braziliensis contains 52 amastin genes belonging to all four previously described amastin subfamilies and that the expression of members of all subfamilies is upregulated in L. braziliensis amastigotes. Although primary sequence alignments showed no homology to any known protein sequence, homology searches based on secondary structure predictions indicate that amastins are related to claudins, a group of proteins that are components of eukaryotic tight junction complexes. By knocking-down the expression of δ-amastins in L. braziliensis, their essential role during infection became evident. δ-amastin knockdown parasites showed impaired growth after in vitro infection of mouse macrophages and completely failed to produce infection when inoculated in BALB/c mice, an attenuated phenotype that was reverted by the re-expression of an RNAi-resistant amastin gene. Further highlighting their essential role in host-parasite interactions, electron microscopy analyses of macrophages infected with amastin knockdown parasites showed significant alterations in the tight contact that is normally observed between the surface of wild type amastigotes and the membrane of the parasitophorous vacuole.

Comparative Genomics of Interreplichore Translocations in Bacteria: A Measure of Chromosome Topology?

PubMed

Khedkar, Supriya; Seshasayee, Aswin Sai Narain

2016-06-01

Genomes evolve not only in base sequence but also in terms of their architecture, defined by gene organization and chromosome topology. Whereas genome sequence data inform us about the changes in base sequences for a large variety of organisms, the study of chromosome topology is restricted to a few model organisms studied using microscopy and chromosome conformation capture techniques. Here, we exploit whole genome sequence data to study the link between gene organization and chromosome topology in bacteria. Using comparative genomics across ∼250 pairs of closely related bacteria we show that: (a) many organisms show a high degree of interreplichore translocations throughout the chromosome and not limited to the inversion-prone terminus (ter) or the origin of replication (oriC); (b) translocation maps may reflect chromosome topologies; and (c) symmetric interreplichore translocations do not disrupt the distance of a gene from oriC or affect gene expression states or strand biases in gene densities. In summary, we suggest that translocation maps might be a first line in defining a gross chromosome topology given a pair of closely related genome sequences. Copyright © 2016 Khedkar and Seshasayee.

Comparative Genomics of Interreplichore Translocations in Bacteria: A Measure of Chromosome Topology?

PubMed Central

Khedkar, Supriya; Seshasayee, Aswin Sai Narain

2016-01-01

Genomes evolve not only in base sequence but also in terms of their architecture, defined by gene organization and chromosome topology. Whereas genome sequence data inform us about the changes in base sequences for a large variety of organisms, the study of chromosome topology is restricted to a few model organisms studied using microscopy and chromosome conformation capture techniques. Here, we exploit whole genome sequence data to study the link between gene organization and chromosome topology in bacteria. Using comparative genomics across ∼250 pairs of closely related bacteria we show that: (a) many organisms show a high degree of interreplichore translocations throughout the chromosome and not limited to the inversion-prone terminus (ter) or the origin of replication (oriC); (b) translocation maps may reflect chromosome topologies; and (c) symmetric interreplichore translocations do not disrupt the distance of a gene from oriC or affect gene expression states or strand biases in gene densities. In summary, we suggest that translocation maps might be a first line in defining a gross chromosome topology given a pair of closely related genome sequences. PMID:27172194

Characterization of protein-protein interaction domains within the baculovirus Autographa californica multiple nucleopolyhedrovirus late expression factor LEF-3.

PubMed

Downie, Kelsey; Adetola, Gbolagade; Carstens, Eric B

2013-11-01

Autographa californica nucleopolyhedrovirus late expression factor 3 (LEF-3) is required for late viral gene expression probably through its numerous functions related to DNA replication, including nuclear localization of the virus helicase P143 and binding to ssDNA. LEF-3 appears to interact with itself as a homo-oligomer, although the details of this oligomeric structure are not yet known. To examine LEF-3-LEF-3 interactions, a bimolecular fluorescent protein complementation assay was used. Pairs of recombinant plasmids expressing full-length LEF-3 fused to one of two complementary fragments (V1 or V2) of a variant of yellow fluorescent protein named 'Venus' were constructed. Plasmids expressing fusions with complementary fragments of Venus were co-transfected into Sf21 cells and analysed by fluorescence microscopy. Co-transfected plasmids expressing full-length V1-LEF-3 and V2-LEF-3 showed positive fluorescence, confirming the formation of homo-oligomers. A series of truncated V1/V2-LEF-3 fusions was constructed and used to investigate interactions with one another as well as with full-length LEF-3.

RNA interference-mediated NOTCH3 knockdown induces phenotype switching of vascular smooth muscle cells in vitro

PubMed Central

Liu, Nan; Li, Ying; Chen, Hui; Wei, Wei; An, Yulin; Zhu, Guangming

2015-01-01

Notch3 plays an important role in differentiation, migration and signal transduction of vascular smooth muscle cells (VSMCs). In this study, we used RNA interference (RNAi) technique to investigate the effect of knocking down the expression of the NOTCH3 gene in VSMCs on the phenotype determination under pathologic status. Real-time PCR and Western Blot experiments verified the expression levels of Notch3 mRNA and protein were reduced more than 40% and 50% in the NOTCH3 siRNA group. When the expression of Notch3 was decreased, the proliferation, apoptosis and immigration of VSMCs were enhanced compared to control groups (P < 0.01). NOTCH3 siRNA VSMCs observed using confocal microscopy showed abnormal nuclear configuration, a disorganized actin filament system, polygonal cell shapes, and decreasing cell sizes. Additionally, knocking down the expression of NOTCH3 may evoke the CASR and FAK expression. In Conclusion, interfering with the expression of NOTCH3 causes VSMCs to exhibit an intermediate phenotype. CaSR and FAK may be involved in the Notch3 signaling pathway. PMID:26550181

RNA interference-mediated NOTCH3 knockdown induces phenotype switching of vascular smooth muscle cells in vitro.

PubMed

Liu, Nan; Li, Ying; Chen, Hui; Wei, Wei; An, Yulin; Zhu, Guangming

2015-01-01

Notch3 plays an important role in differentiation, migration and signal transduction of vascular smooth muscle cells (VSMCs). In this study, we used RNA interference (RNAi) technique to investigate the effect of knocking down the expression of the NOTCH3 gene in VSMCs on the phenotype determination under pathologic status. Real-time PCR and Western Blot experiments verified the expression levels of Notch3 mRNA and protein were reduced more than 40% and 50% in the NOTCH3 siRNA group. When the expression of Notch3 was decreased, the proliferation, apoptosis and immigration of VSMCs were enhanced compared to control groups (P < 0.01). NOTCH3 siRNA VSMCs observed using confocal microscopy showed abnormal nuclear configuration, a disorganized actin filament system, polygonal cell shapes, and decreasing cell sizes. Additionally, knocking down the expression of NOTCH3 may evoke the CASR and FAK expression. In Conclusion, interfering with the expression of NOTCH3 causes VSMCs to exhibit an intermediate phenotype. CaSR and FAK may be involved in the Notch3 signaling pathway.

Btk inhibition suppresses agonist-induced human macrophage activation and inflammatory gene expression in RA synovial tissue explants.

PubMed

Hartkamp, Linda M; Fine, Jay S; van Es, Inge E; Tang, Man Wai; Smith, Michael; Woods, John; Narula, Satwant; DeMartino, Julie; Tak, Paul P; Reedquist, Kris A

2015-08-01

Bruton's tyrosine kinase (Btk) is required for B lymphocyte and myeloid cell contributions to pathology in murine models of arthritis. Here, we examined the potential contributions of synovial Btk expression and activation to inflammation in rheumatoid arthritis (RA). Btk was detected by immunohistochemistry and digital image analysis in synovial tissue from biologically naive RA (n=16) and psoriatic arthritis (PsA) (n=12) patients. Cell populations expressing Btk were identified by immunofluorescent double labelling confocal microscopy, quantitative (q-) PCR and immunoblotting. The effects of a Btk-specific inhibitor, RN486, on gene expression in human macrophages and RA synovial tissue explants (n=8) were assessed by qPCR, ELISA and single-plex assays. Btk was expressed at equivalent levels in RA and PsA synovial tissue, restricted to B lymphocytes, monocytes, macrophages and mast cells. RN486 significantly inhibited macrophage IL-6 production induced by Fc receptor and CD40 ligation. RN486 also reduced mRNA expression of overlapping gene sets induced by IgG, CD40 ligand (CD40L) and RA synovial fluid, and significantly suppressed macrophage production of CD40L-induced IL-8, TNF, MMP-1 and MMP-10, LPS-induced MMP-1, MMP-7 and MMP-10 production, and spontaneous production of IL-6, PDGF, CXCL-9 and MMP-1 by RA synovial explants. Btk is expressed equivalently in RA and PsA synovial tissue, primarily in macrophages. Btk activity is needed to drive macrophage activation in response to multiple agonists relevant to inflammatory arthritis, and promotes RA synovial tissue cytokine and MMP production. Pharmacological targeting of Btk may be of therapeutic benefit in the treatment of RA and other inflammatory diseases. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Autographa californica multiple nucleopolyhedrovirus ac53 plays a role in nucleocapsid assembly

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

Liu Chao; Li Zhaofei; Wu Wenbi

2008-12-05

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf53 (ac53) is a highly conserved gene existing in all sequenced Lepidoptera and Hymenoptera baculoviruses, but its function remains unknown. To investigate its role in the baculovirus life cycle, an ac53 deletion virus (vAc{sup ac53KO-PH-GFP}) was generated through homologous recombination in Escherichia coli. Fluorescence and light microscopy and titration analysis revealed that vAc{sup ac53KO-PH-GFP} could not produce infectious budded virus in infected Sf9 cells. Real-time PCR demonstrated that the ac53 deletion did not affect the levels of viral DNA replication. Electron microscopy showed that many lucent tubular shells devoid of the nucleoprotein core are presentmore » in the virogenic stroma and ring zone, indicating that the ac53 knockout affected nucleocapsid assembly. With a recombinant virus expressing an Ac53-GFP fusion protein, we observed that Ac53 was distributed within the cytoplasm and nucleus at 24 h post-infection, but afterwards accumulated predominantly near the nucleus-cytoplasm boundary. These data demonstrate that ac53 is involved in nucleocapsid assembly and is an essential gene for virus production.« less

The Ras effector RASSF2 is a novel tumor-suppressor gene in human colorectal cancer.

PubMed

Akino, Kimishige; Toyota, Minoru; Suzuki, Hiromu; Mita, Hiroaki; Sasaki, Yasushi; Ohe-Toyota, Mutsumi; Issa, Jean-Pierre J; Hinoda, Yuji; Imai, Kohzoh; Tokino, Takashi

2005-07-01

Activation of Ras signaling is a hallmark of colorectal cancer (CRC), but the roles of negative regulators of Ras are not fully understood. Our aim was to address that question by surveying genetic and epigenetic alterations of Ras-Ras effector genes in CRC cells. The expression and methylation status of 6 RASSF family genes were examined using RT-PCR and bisulfite PCR in CRC cell lines and in primary CRCs and colorectal adenomas. Colony formation assays and flow cytometry were used to assess the tumor suppressor activities of RASSF1 and RASSF2. Immunofluorescence microscopy was used to determine the effect of altered RASSF2 expression on cell morphology. Mutations of K- ras , BRAF, and p53 were identified using single-strand conformation analysis and direct sequencing. Aberrant methylation and histone deacetylation of RASSF2 was associated with the gene's silencing in CRC. The activities of RASSF2, which were distinct from those of RASSF1, included induction of morphologic changes and apoptosis; moreover, its ability to prevent cell transformation suggests that RASSF2 acts as a tumor suppressor in CRC. Primary CRCs that showed K- ras /BRAF mutations also frequently showed RASSF2 methylation, and inactivation of RASSF2 enhanced K- ras -induced oncogenic transformation. RASSF2 methylation was also frequently identified in colorectal adenomas. RASSF2 is a novel tumor suppressor gene that regulates Ras signaling and plays a pivotal role in the early stages of colorectal tumorigenesis.

High ordered biomineralization induced by carbon nanoparticles in the sea urchin Paracentrotus lividus.

PubMed

Manno, Daniela; Carata, Elisabetta; Tenuzzo, Bernadetta A; Panzarini, Elisa; Buccolieri, Alessandro; Filippo, Emanuela; Rossi, Marco; Serra, Antonio; Dini, Luciana

2012-12-14

A surprising and unexpected biomineralization process was observed during toxicological assessment of carbon nanoparticles on Paracentrotus lividus (sea urchin) pluteus larvae. The larvae activate a process of defense against external material, by incorporating the nanoparticles into microstructures of aragonite similarly to pearl oysters. Aiming at a better understanding of this phenomenon, the larvae were exposed to increasing concentrations of carbon nanoparticles and the biomineralization products were analyzed by electron microscopy, x-ray diffraction and Raman spectroscopy. In order to evaluate the possible influence of Sp-CyP-1 expression on this biomineralization process by larvae, analyses of gene expression (Sp-CyP-1) and calcein labeling were performed. Overall, we report experimental evidence about the capability of carbon nanoparticles to induce an increment of Sp-CyP-1 expression with the consequent activation of a biomineralization process leading to the production of a new pearl-like biomaterial never previously observed in sea urchins.

High ordered biomineralization induced by carbon nanoparticles in the sea urchin Paracentrotus lividus

NASA Astrophysics Data System (ADS)

Manno, Daniela; Carata, Elisabetta; Tenuzzo, Bernadetta A.; Panzarini, Elisa; Buccolieri, Alessandro; Filippo, Emanuela; Rossi, Marco; Serra, Antonio; Dini, Luciana

2012-12-01

A surprising and unexpected biomineralization process was observed during toxicological assessment of carbon nanoparticles on Paracentrotus lividus (sea urchin) pluteus larvae. The larvae activate a process of defense against external material, by incorporating the nanoparticles into microstructures of aragonite similarly to pearl oysters. Aiming at a better understanding of this phenomenon, the larvae were exposed to increasing concentrations of carbon nanoparticles and the biomineralization products were analyzed by electron microscopy, x-ray diffraction and Raman spectroscopy. In order to evaluate the possible influence of Sp-CyP-1 expression on this biomineralization process by larvae, analyses of gene expression (Sp-CyP-1) and calcein labeling were performed. Overall, we report experimental evidence about the capability of carbon nanoparticles to induce an increment of Sp-CyP-1 expression with the consequent activation of a biomineralization process leading to the production of a new pearl-like biomaterial never previously observed in sea urchins.

Supported Lipid Bilayer Technology for the Study of Cellular Interfaces

PubMed Central

Crites, Travis J.; Maddox, Michael; Padhan, Kartika; Muller, James; Eigsti, Calvin; Varma, Rajat

2015-01-01

Glass-supported lipid bilayers presenting freely diffusing proteins have served as a powerful tool for studying cell-cell interfaces, in particular, T cell–antigen presenting cell (APC) interactions, using optical microscopy. Here we expand upon existing protocols and describe the preparation of liposomes by an extrusion method, and describe how this system can be used to study immune synapse formation by Jurkat cells. We also present a method for forming such lipid bilayers on silica beads for the study of signaling responses by population methods, such as western blotting, flow cytometry, and gene-expression analysis. Finally, we describe how to design and prepare transmembrane-anchored protein-laden liposomes, following expression in suspension CHO (CHOs) cells, a mammalian expression system alternative to insect and bacterial cell lines, which do not produce mammalian glycosylation patterns. Such transmembrane-anchored proteins may have many novel applications in cell biology and immunology. PMID:26331983

Temporal Characterization of Microglia/Macrophage Phenotypes in a Mouse Model of Neonatal Hypoxic-Ischemic Brain Injury

PubMed Central

Hellström Erkenstam, Nina; Smith, Peter L. P.; Fleiss, Bobbi; Nair, Syam; Svedin, Pernilla; Wang, Wei; Boström, Martina; Gressens, Pierre; Hagberg, Henrik; Brown, Kelly L.; Sävman, Karin; Mallard, Carina

2016-01-01

Immune cells display a high degree of phenotypic plasticity, which may facilitate their participation in both the progression and resolution of injury-induced inflammation. The purpose of this study was to investigate the temporal expression of genes associated with classical and alternative polarization phenotypes described for macrophages and to identify related cell populations in the brain following neonatal hypoxia-ischemia (HI). HI was induced in 9-day old mice and brain tissue was collected up to 7 days post-insult to investigate expression of genes associated with macrophage activation. Using cell-markers, CD86 (classic activation) and CD206 (alternative activation), we assessed temporal changes of CD11b+ cell populations in the brain and studied the protein expression of the immunomodulatory factor galectin-3 in these cells. HI induced a rapid regulation (6 h) of genes associated with both classical and alternative polarization phenotypes in the injured hemisphere. FACS analysis showed a marked increase in the number of CD11b+CD86+ cells at 24 h after HI (+3667%), which was coupled with a relative suppression of CD11b+CD206+ cells and cells that did not express neither CD86 nor CD206. The CD11b+CD206+ population was mixed with some cells also expressing CD86. Confocal microscopy confirmed that a subset of cells expressed both CD86 and CD206, particularly in injured gray and white matter. Protein concentration of galectin-3 was markedly increased mainly in the cell population lacking CD86 or CD206 in the injured hemisphere. These cells were predominantly resident microglia as very few galectin-3 positive cells co-localized with infiltrating myeloid cells in Lys-EGFP-ki mice after HI. In summary, HI was characterized by an early mixed gene response, but with a large expansion of mainly the CD86 positive population during the first day. However, the injured hemisphere also contained a subset of cells expressing both CD86 and CD206 and a large population that expressed neither activation marker CD86 nor CD206. Interestingly, these cells expressed the highest levels of galectin-3 and were found to be predominantly resident microglia. Galectin-3 is a protein involved in chemotaxis and macrophage polarization suggesting a novel role in cell infiltration and immunomodulation for this cell population after neonatal injury. PMID:28018179

Decreased Rac1 Cardiac Expression in Nitrofen-Induced Diaphragmatic Hernia.

PubMed

Nakamura, Hiroki; Zimmer, Julia; Puri, Prem

2018-02-01

 The high incidence of cardiac malformations in humans and animal models with congenital diaphragmatic hernia (CDH) is well known. The hypoplasia of left heart is common among fetuses with CDH and has been identified as a poor prognostic factor. However, the precise mechanisms underlying cardiac maldevelopment in CDH are not fully understood. Ras-related C3 botulinum toxin substrate 1 (Rac1) plays a key role in cardiomyocyte polarity and embryonic heart development. Deficiency of Rac1 is reported to impair elongation and cytoskeletal organization of cardiomyocytes, resulting in congenital cardiac defects. We designed this study to test the hypothesis that Rac1 expression is downregulated in the developing hearts of rats with nitrofen-induced CDH.  Following ethical approval (REC1103), time-pregnant Sprague Dawley rats received nitrofen or vehicle on gestational day 9 (D9). Fetuses were sacrificed on D18 and D21 and divided into CDH and control (CTRL) ( n  = 6 for each group and time point). Quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting, and confocal-immunofluorescence microscopy were performed to detect cardiac gene and protein expression of Rac1.  qRT-PCR and Western blot analysis revealed that Rac1 expression was significantly decreased in the CDH group compared with controls ( p  < 0.05). Confocal-immunofluorescence microscopy revealed that Rac1 cardiac expression was markedly decreased in the CDH group compared with controls.  Decreased cardiac Rac1 expression in the nitrofen-induced CDH suggests that Rac1 deficiency during morphogenesis may impair structural cardiac remodeling, resulting in congenital cardiac defects. Georg Thieme Verlag KG Stuttgart · New York.

Impact of traffic-related air pollution on the expression of Platanus orientalis pollen allergens.

PubMed

Sedghy, Farnaz; Sankian, Mojtaba; Moghadam, Maliheh; Ghasemi, Ziba; Mahmoudi, Mahmoud; Varasteh, Abdol-Reza

2017-01-01

Air pollutants and their interaction with environmental allergens have been considered as an important reason for the recent increase in the prevalence of allergic diseases. The aim of this study was to investigate the traffic pollution effect, as a stressor, on Platanus orientalis pollen allergens messenger RNA (mRNA) and protein expression. P. orientalis pollen grains were collected along main streets of heavy traffic and from unpolluted sites in Mashhad city, in northeast Iran. The pollen samples were examined by scanning electron microscopy. To assess the abundance of pollen allergens (Pla or 1, Pla or 2, and Pla or 3) from polluted and unpolluted sites, immunoblotting was performed. Moreover, the sequences encoding P. orientalis allergens were amplified using real-time PCR. Scanning electron microscopy showed a number of particles of 150-550 nm on the surface of pollen from polluted sites. Also, protein and gene expression levels of Pla or 1 and Pla or 3 were considerably greater in pollen samples from highly polluted areas than in pollen from unpolluted areas (p < 0.05). In contrast, no statically significant difference in Pla or 2 protein and mRNA expression level was found between samples from the two areas. We found greater expression of allergens involved in plant defense mechanisms (Pla or 1 and Pla or 3) in polluted sites than in unpolluted ones. The high expression of these proteins can lead to an increase in the prevalence of allergic diseases. These findings suggest the necessity of supporting public policies aimed at controlling traffic pollution to improve air quality and prevent the subsequent clinical outcomes and new cases of asthma.

Enhanced differentiation of human embryonic stem cells into cardiomyocytes by combining hanging drop culture and 5-azacytidine treatment.

PubMed

Yoon, Byung Sun; Yoo, Seung Jun; Lee, Jeoung Eun; You, Seungkwon; Lee, Hoon Taek; Yoon, Hyun Soo

2006-04-01

Cell replacement therapy is a promising approach for the treatment of cardiac diseases. It is, however, challenged by a limited supply of appropriate cells. Therefore, we have investigated whether functional cardiomyocytes can be efficiently generated from human embryonic stem cells (hESCs). In this study, we developed an efficient protocol for the generation of functional cardiomyocytes from hESCs by combining hanging drop culture and 5-azacytidine, a well-known demethylating agent, and then evaluated the expression of cardiac-specific markers. hESCs were cultured both in the medium without or with 0.1, 1, or 10 microM of 5-azacytidine under a hanging drop culture. The expression of several cardiac-specific markers was determined by real-time PCR, RT-PCR, immunofluorescence, and confocal microscopy. To verify the structural and functional properties of hESC-derived cardiomyocytes, we performed electron microscopy and electrophysiological recording. The efficiency of beating cell generation was significantly improved in the hanging drop culture compared with that in suspension culture. Treatment of hESCs with 0.1 microM of 5-azacytidine for 1-3 days significantly increased the number of beating cells and simultaneously enhanced the expression of cardiac-specific markers. Transmission electron microscopy and electrophysiological recording showed that hESC-derived cardiomyocytes acquired structural and functional properties of cardiomyocytes. In conclusion, these results suggest that differentiation of hESCs into cardiomyocytes can be enhanced by the combination of hanging drop culture and 5-azacytidine treatment. Also the methylation status of genes related to cardiomyocyte development may play an important role in the differentiation of hESCs into cardiomyocytes.

Curli Fibers Are Highly Conserved between Salmonella typhimurium and Escherichia coli with Respect to Operon Structure and Regulation

PubMed Central

Römling, Ute; Bian, Zhao; Hammar, Mårten; Sierralta, Walter D.; Normark, Staffan

1998-01-01

Mouse-virulent Salmonella typhimurium strains SR-11 and ATCC 14028-1s express curli fibers, thin aggregative fibers, at ambient temperature on plates as judged by Western blot analysis and electron microscopy. Concomitantly with curli expression, cells develop a rough and dry colony morphology and bind the dye Congo red (called the rdar morphotype). Cloning and characterization of the two divergently transcribed operons required for curli biogenesis, csgBA(C) and csgDEFG, from S. typhimurium SR-11 revealed the same gene order and flanking genes as in Escherichia coli. The divergence of the curli region between S. typhimurium and E. coli at the nucleotide level is above average (22.4%). However, a high level of conservation at the protein level, which ranged from 86% amino acid homology for the fiber subunit CsgA to 99% homology for the lipoprotein CsgG, implies functional constraints on the gene products. Consequently, S. typhimurium genes on low-copy-number plasmids were able to complement respective E. coli mutants, although not always to wild-type levels. rpoS and ompR are required for transcriptional activation of (at least) the csgD promoter. The high degree of conservation at the protein level and the identical regulation patterns in E. coli and S. typhimurium suggest similar roles of curli fibers in the same ecological niche in the two species. PMID:9457880

Constitutive expression of ftsZ overrides the whi developmental genes to initiate sporulation of Streptomyces coelicolor.

PubMed

Willemse, Joost; Mommaas, A Mieke; van Wezel, Gilles P

2012-03-01

The filamentous soil bacteria Streptomyces undergo a highly complex developmental programme. Before streptomycetes commit themselves to sporulation, distinct morphological checkpoints are passed in the aerial hyphae that are subject to multi-level control by the whi sporulation genes. Here we show that whi-independent expression of FtsZ restores sporulation to the early sporulation mutants whiA, whiB, whiG, whiH, whiI and whiJ. Viability, stress resistance and high-resolution electron microscopy underlined that viable spores were formed. However, spores from sporulation-restored whiA and whiG mutants showed defects in DNA segregation/condensation, while spores from the complemented whiB mutant had increased stress sensitivity, perhaps as a result of changes in the spore sheath. In contrast to the whi mutants, normal sporulation of ssgB null mutants-which fail to properly localise FtsZ-could not be restored by enhancing FtsZ protein levels, forming spore-like bodies that lack spore walls. Our data strongly suggest that the whi genes control a decisive event towards sporulation of streptomycetes, namely the correct timing of developmental ftsZ transcription. The biological significance may be to ensure that sporulation-specific cell division will only start once sufficient aerial mycelium biomass has been generated. Our data shed new light on the longstanding question as to how whi genes control sporulation, which has intrigued scientists for four decades.

Polyhedron-like inclusion body formation by a mutant nucleopolyhedrovirus expressing the granulin gene from a granulovirus.

PubMed

Zhou, C E; Ko, R; Maeda, S

1998-01-20

The polyhedrin gene in Bombyx mori nucleopolyhedrovirus (BmNPV) was replaced with the granulin gene of Trichoplusia ni granulovirus (TnGV). The substitution was verified by Southern hybridization, and expression of granulin by the mutant virus, BmGran, was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by amino acid sequencing of the predominant protein of BmGran inclusion bodies (IBs). Light and electron microscopy examination of BmGran-infected B. mori and BmN cells revealed large, cuboidal, polyhedron-like IBs in the nucleus and cytoplasm, but granules were not seen. IBs contained small, parallel, electron-dense streaks, which defined the geometric pattern of crystallization. Geometric patterns of nuclear IBs were frequently disrupted by occlusion of polyhedron envelope fragments, resulting in IB instability and fracturing. Virions were not embedded in most of the polyhedron-like IBs, but accumulated with polyhedron envelope fragments. Some virions were coated with matrix protein and were partially wrapped by polyhedron envelope. These results suggested that (1) the amino acid sequence of granulin insufficient for determining IB morphology in TnGV-infected cells, and TnGV may have genes, not present in BmNPV, that control granule formation, and (2) interactions among the virion, the IB envelope, and the matrix protein may be important in virion occlusion and IB morphology and stability.

Antisense oligodeoxynucleotide to the cystic fibrosis gene inhibits anion transport in normal cultured sweat duct cells

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

Sorscher, E.J.; Kirk, K.L.; Weaver, M.L.

The authors have tested the hypothesis that the cystic fibrosis (CF) gene product, called the CF transmembrane conductance regulator (CFTR), mediates anion transport in normal human sweat duct cells. Sweat duct cells in primary culture were treated with oligodeoxynucleotides that were antisense to the CFTR gene transcript in order to block the expression of the wild-type CFTR. Anion transport in CFTR transcript antisense-treated cells was then assessed with a halide-specific dye, 6-methoxy-N-(3-sulfopropryl)quinolinium, and fluorescent digital imaging microscopy to monitor halide influx and efflux from single sweat duct cells. Antisense oligodeoxynucleotide treatment for 24 hr virtually abolished Cl{sup {minus}} transport inmore » sweat duct cells compared with untreated cells or control cells treated with sense oligodeoxynucleotides. Br{sup {minus}} uptake into sweat duct cells was also blocked after a 24-hr CFTR transcript antisense treatments, but not after treatments for only 4 hr. Lower concentrations of antisense oligodeoxynucleotides were less effective at inhibiting Cl{sup {minus}} transport. These results indicate that oligodeoxynucleotides that are antisense to CFTR transcript inhibit sweat duct Cl{sup {minus}} permeability in both a time-dependent and dose-dependent manner. This approach provides evidence that inhibition of the expression of the wild-type CFTR gene in a normal, untransfected epithelial cell results in an inhibition of Cl{sup {minus}} permeability.« less

Role of Aspergillus niger acrA in Arsenic Resistance and Its Use as the Basis for an Arsenic Biosensor

PubMed Central

Choe, Se-In; Gravelat, Fabrice N.; Al Abdallah, Qusai; Lee, Mark J.; Gibbs, Bernard F.

2012-01-01

Arsenic contamination of groundwater sources is a major issue worldwide, since exposure to high levels of arsenic has been linked to a variety of health problems. Effective methods of detection are thus greatly needed as preventive measures. In an effort to develop a fungal biosensor for arsenic, we first identified seven putative arsenic metabolism and transport genes in Aspergillus niger, a widely used industrial organism that is generally regarded as safe (GRAS). Among the genes tested for RNA expression in response to arsenate, acrA, encoding a putative plasma membrane arsenite efflux pump, displayed an over 200-fold increase in gene expression in response to arsenate. We characterized the function of this A. niger protein in arsenic efflux by gene knockout and confirmed that AcrA was located at the cell membrane using an enhanced green fluorescent protein (eGFP) fusion construct. Based on our observations, we developed a putative biosensor strain containing a construct of the native promoter of acrA fused with egfp. We analyzed the fluorescence of this biosensor strain in the presence of arsenic using confocal microscopy and spectrofluorimetry. The biosensor strain reliably detected both arsenite and arsenate in the range of 1.8 to 180 μg/liter, which encompasses the threshold concentrations for drinking water set by the World Health Organization (10 and 50 μg/liter). PMID:22467499

(Machine-)Learning to analyze in vivo microscopy: Support vector machines.

PubMed

Wang, Michael F Z; Fernandez-Gonzalez, Rodrigo

2017-11-01

The development of new microscopy techniques for super-resolved, long-term monitoring of cellular and subcellular dynamics in living organisms is revealing new fundamental aspects of tissue development and repair. However, new microscopy approaches present several challenges. In addition to unprecedented requirements for data storage, the analysis of high resolution, time-lapse images is too complex to be done manually. Machine learning techniques are ideally suited for the (semi-)automated analysis of multidimensional image data. In particular, support vector machines (SVMs), have emerged as an efficient method to analyze microscopy images obtained from animals. Here, we discuss the use of SVMs to analyze in vivo microscopy data. We introduce the mathematical framework behind SVMs, and we describe the metrics used by SVMs and other machine learning approaches to classify image data. We discuss the influence of different SVM parameters in the context of an algorithm for cell segmentation and tracking. Finally, we describe how the application of SVMs has been critical to study protein localization in yeast screens, for lineage tracing in C. elegans, or to determine the developmental stage of Drosophila embryos to investigate gene expression dynamics. We propose that SVMs will become central tools in the analysis of the complex image data that novel microscopy modalities have made possible. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman. Copyright © 2017 Elsevier B.V. All rights reserved.

Localization microscopy of DNA in situ using Vybrant(®) DyeCycle™ Violet fluorescent probe: A new approach to study nuclear nanostructure at single molecule resolution.

PubMed

Żurek-Biesiada, Dominika; Szczurek, Aleksander T; Prakash, Kirti; Mohana, Giriram K; Lee, Hyun-Keun; Roignant, Jean-Yves; Birk, Udo J; Dobrucki, Jurek W; Cremer, Christoph

2016-05-01

Higher order chromatin structure is not only required to compact and spatially arrange long chromatids within a nucleus, but have also important functional roles, including control of gene expression and DNA processing. However, studies of chromatin nanostructures cannot be performed using conventional widefield and confocal microscopy because of the limited optical resolution. Various methods of superresolution microscopy have been described to overcome this difficulty, like structured illumination and single molecule localization microscopy. We report here that the standard DNA dye Vybrant(®) DyeCycle™ Violet can be used to provide single molecule localization microscopy (SMLM) images of DNA in nuclei of fixed mammalian cells. This SMLM method enabled optical isolation and localization of large numbers of DNA-bound molecules, usually in excess of 10(6) signals in one cell nucleus. The technique yielded high-quality images of nuclear DNA density, revealing subdiffraction chromatin structures of the size in the order of 100nm; the interchromatin compartment was visualized at unprecedented optical resolution. The approach offers several advantages over previously described high resolution DNA imaging methods, including high specificity, an ability to record images using a single wavelength excitation, and a higher density of single molecule signals than reported in previous SMLM studies. The method is compatible with DNA/multicolor SMLM imaging which employs simple staining methods suited also for conventional optical microscopy. Copyright © 2016. Published by Elsevier Inc.

Characterization of the Bm61 of the Bombyx mori nucleopolyhedrovirus.

PubMed

Shen, Hongxing; Chen, Keping; Yao, Qin; Zhou, Yang

2009-07-01

orf61 (bm61) of Bombyx mori Nucleopolyhedrovirus (BmNPV) is a highly conserved baculovirus gene, suggesting that it performs an important role in the virus life cycle whose function is unknown. In this study, we describe the characterization of bm61. Quantitative polymerase chain reaction (qPCR) and western blot analysis demonstrated that bm61 was expressed as a late gene. Immunofluorescence analysis by confocal microscopy showed that BM61 protein was localized on nuclear membrane and in intranuclear ring zone of infected cells. Structure localization of the BM61 in BV and ODV by western analysis demonstrated that BM61 was the protein of both BV and ODV. In addition, our data indicated that BM61 was a late structure protein localized in nucleus.

Fusions between green fluorescent protein and beta-glucuronidase as sensitive and vital bifunctional reporters in plants.

PubMed

Quaedvlieg, N E; Schlaman, H R; Admiraal, P C; Wijting, S E; Stougaard, J; Spaink, H P

1998-07-01

By fusing the genes encoding green fluorescent protein (GFP) and beta-glucuronidase (GUS) we have created a set of bifunctional reporter constructs which are optimized for use in transient and stable expression studies in plants. This approach makes it possible to combine the advantage of GUS, its high sensitivity in histochemical staining, with the advantages of GFP as a vital marker. The fusion proteins were functional in transient expression studies in tobacco using either DNA bombardment or potato virus X as a vector, and in stably transformed Arabidopsis thaliana and Lotus japonicus plants. The results show that high level of expression does not interfere with efficient stable transformation in A. thaliana and L. japonicus. Using confocal laser scanning microscopy we show that the fusion constructs are very suitable for promoter expression studies in all organs of living plants, including root nodules. The use of these reporter constructs in the model legume L. japonicus offers exciting new possibilities for the study of the root nodulation process.

Fusions between green fluorescent protein and beta-glucuronidase as sensitive and vital bifunctional reporters in plants.

PubMed

Quaedvlieg, N E; Schlaman, H R; Admiraal, P C; Wijting, S E; Stougaard, J; Spaink, H P

1998-11-01

By fusing the genes encoding green fluorescent protein (GFP) and beta-glucuronidase (GUS) we have created a set of bifunctional reporter constructs which are optimized for use in transient and stable expression studies in plants. This approach makes it possible to combine the advantage of GUS, its high sensitivity in histochemical staining, with the advantages of GFP as a vital marker. The fusion proteins were functional in transient expression studies in tobacco using either DNA bombardment or potato virus X as a vector, and in stably transformed Arabidopsis thaliana and Lotus japonicus plants. The results show that high level of expression does not interfere with efficient stable transformation in A. thaliana and L. japonicus. Using confocal laser scanning microscopy we show that the fusion constructs are very suitable for promoter expression studies in all organs of living plants, including root nodules. The use of these reporter constructs in the model legume L. japonicus offers exciting new possibilities for the study of the root nodulation process.

Wongabel rhabdovirus accessory protein U3 targets the SWI/SNF chromatin remodeling complex.

PubMed

Joubert, D Albert; Rodriguez-Andres, Julio; Monaghan, Paul; Cummins, Michelle; McKinstry, William J; Paradkar, Prasad N; Moseley, Gregory W; Walker, Peter J

2015-01-15

Wongabel virus (WONV) is an arthropod-borne rhabdovirus that infects birds. It is one of the growing array of rhabdoviruses with complex genomes that encode multiple accessory proteins of unknown function. In addition to the five canonical rhabdovirus structural protein genes (N, P, M, G, and L), the 13.2-kb negative-sense single-stranded RNA (ssRNA) WONV genome contains five uncharacterized accessory genes, one overlapping the N gene (Nx or U4), three located between the P and M genes (U1 to U3), and a fifth one overlapping the G gene (Gx or U5). Here we show that WONV U3 is expressed during infection in insect and mammalian cells and is required for efficient viral replication. A yeast two-hybrid screen against a mosquito cell cDNA library identified that WONV U3 interacts with the 83-amino-acid (aa) C-terminal domain of SNF5, a component of the SWI/SNF chromatin remodeling complex. The interaction was confirmed by affinity chromatography, and nuclear colocalization was established by confocal microscopy. Gene expression studies showed that SNF5 transcripts are upregulated during infection of mosquito cells with WONV, as well as West Nile virus (Flaviviridae) and bovine ephemeral fever virus (Rhabdoviridae), and that SNF5 knockdown results in increased WONV replication. WONV U3 also inhibits SNF5-regulated expression of the cytokine gene CSF1. The data suggest that WONV U3 targets the SWI/SNF complex to block the host response to infection. The rhabdoviruses comprise a large family of RNA viruses infecting plants, vertebrates, and invertebrates. In addition to the major structural proteins (N, P, M, G, and L), many rhabdoviruses encode a diverse array of accessory proteins of largely unknown function. Understanding the role of these proteins may reveal much about host-pathogen interactions in infected cells. Here we examine accessory protein U3 of Wongabel virus, an arthropod-borne rhabdovirus that infects birds. We show that U3 enters the nucleus and interacts with SNF5, a component of the chromatin remodeling complex that is upregulated in response to infection and restricts viral replication. We also show that U3 inhibits SNF5-regulated expression of the cytokine colony-stimulating factor 1 (CSF1), suggesting that it targets the chromatin remodeling complex to block the host response to infection. This study appears to provide the first evidence of a virus targeting SNF5 to inhibit host gene expression. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Wongabel Rhabdovirus Accessory Protein U3 Targets the SWI/SNF Chromatin Remodeling Complex

PubMed Central

Joubert, D. Albert; Rodriguez-Andres, Julio; Monaghan, Paul; Cummins, Michelle; McKinstry, William J.; Paradkar, Prasad N.; Moseley, Gregory W.

2014-01-01

ABSTRACT Wongabel virus (WONV) is an arthropod-borne rhabdovirus that infects birds. It is one of the growing array of rhabdoviruses with complex genomes that encode multiple accessory proteins of unknown function. In addition to the five canonical rhabdovirus structural protein genes (N, P, M, G, and L), the 13.2-kb negative-sense single-stranded RNA (ssRNA) WONV genome contains five uncharacterized accessory genes, one overlapping the N gene (Nx or U4), three located between the P and M genes (U1 to U3), and a fifth one overlapping the G gene (Gx or U5). Here we show that WONV U3 is expressed during infection in insect and mammalian cells and is required for efficient viral replication. A yeast two-hybrid screen against a mosquito cell cDNA library identified that WONV U3 interacts with the 83-amino-acid (aa) C-terminal domain of SNF5, a component of the SWI/SNF chromatin remodeling complex. The interaction was confirmed by affinity chromatography, and nuclear colocalization was established by confocal microscopy. Gene expression studies showed that SNF5 transcripts are upregulated during infection of mosquito cells with WONV, as well as West Nile virus (Flaviviridae) and bovine ephemeral fever virus (Rhabdoviridae), and that SNF5 knockdown results in increased WONV replication. WONV U3 also inhibits SNF5-regulated expression of the cytokine gene CSF1. The data suggest that WONV U3 targets the SWI/SNF complex to block the host response to infection. IMPORTANCE The rhabdoviruses comprise a large family of RNA viruses infecting plants, vertebrates, and invertebrates. In addition to the major structural proteins (N, P, M, G, and L), many rhabdoviruses encode a diverse array of accessory proteins of largely unknown function. Understanding the role of these proteins may reveal much about host-pathogen interactions in infected cells. Here we examine accessory protein U3 of Wongabel virus, an arthropod-borne rhabdovirus that infects birds. We show that U3 enters the nucleus and interacts with SNF5, a component of the chromatin remodeling complex that is upregulated in response to infection and restricts viral replication. We also show that U3 inhibits SNF5-regulated expression of the cytokine colony-stimulating factor 1 (CSF1), suggesting that it targets the chromatin remodeling complex to block the host response to infection. This study appears to provide the first evidence of a virus targeting SNF5 to inhibit host gene expression. PMID:25392228

Retained Myogenic Potency of Human Satellite Cells from Torn Rotator Cuff Muscles Despite Fatty Infiltration.

PubMed

Koide, Masashi; Hagiwara, Yoshihiro; Tsuchiya, Masahiro; Kanzaki, Makoto; Hatakeyama, Hiroyasu; Tanaka, Yukinori; Minowa, Takashi; Takemura, Taro; Ando, Akira; Sekiguchi, Takuya; Yabe, Yutaka; Itoi, Eiji

2018-01-01

Rotator cuff tears (RCTs) are a common shoulder problem in the elderly that can lead to both muscle atrophy and fatty infiltration due to less physical load. Satellite cells, quiescent cells under the basal lamina of skeletal muscle fibers, play a major role in muscle regeneration. However, the myogenic potency of human satellite cells in muscles with fatty infiltration is unclear due to the difficulty in isolating from small samples, and the mechanism of the progression of fatty infiltration has not been elucidated. The purpose of this study was to analyze the population of myogenic and adipogenic cells in disused supraspinatus (SSP) and intact subscapularis (SSC) muscles of the RCTs from the same patients using fluorescence-activated cell sorting. The microstructure of the muscle with fatty infiltration was observed as a whole mount condition under multi-photon microscopy. Myogenic differentiation potential and gene expression were evaluated in satellite cells. The results showed that the SSP muscle with greater fatty infiltration surrounded by collagen fibers compared with the SSC muscle under multi-photon microscopy. A positive correlation was observed between the ratio of muscle volume to fat volume and the ratio of myogenic precursor to adipogenic precursor. Although no difference was observed in the myogenic potential between the two groups in cell culture, satellite cells in the disused SSP muscle showed higher intrinsic myogenic gene expression than those in the intact SSC muscle. Our results indicate that satellite cells from the disused SSP retain sufficient potential of muscle growth despite the fatty infiltration.

Synaptic vesicle dynamic changes in a model of fragile X.

PubMed

Broek, Jantine A C; Lin, Zhanmin; de Gruiter, H Martijn; van 't Spijker, Heleen; Haasdijk, Elize D; Cox, David; Ozcan, Sureyya; van Cappellen, Gert W A; Houtsmuller, Adriaan B; Willemsen, Rob; de Zeeuw, Chris I; Bahn, Sabine

2016-01-01

Fragile X syndrome (FXS) is a single-gene disorder that is the most common heritable cause of intellectual disability and the most frequent monogenic cause of autism spectrum disorders (ASD). FXS is caused by an expansion of trinucleotide repeats in the promoter region of the fragile X mental retardation gene (Fmr1). This leads to a lack of fragile X mental retardation protein (FMRP), which regulates translation of a wide range of messenger RNAs (mRNAs). The extent of expression level alterations of synaptic proteins affected by FMRP loss and their consequences on synaptic dynamics in FXS has not been fully investigated. Here, we used an Fmr1 knockout (KO) mouse model to investigate the molecular mechanisms underlying FXS by monitoring protein expression changes using shotgun label-free liquid-chromatography mass spectrometry (LC-MS(E)) in brain tissue and synaptosome fractions. FXS-associated candidate proteins were validated using selected reaction monitoring (SRM) in synaptosome fractions for targeted protein quantification. Furthermore, functional alterations in synaptic release and dynamics were evaluated using live-cell imaging, and interpretation of synaptic dynamics differences was investigated using electron microscopy. Key findings relate to altered levels of proteins involved in GABA-signalling, especially in the cerebellum. Further exploration using microscopy studies found reduced synaptic vesicle unloading of hippocampal neurons and increased vesicle unloading in cerebellar neurons, which suggests a general decrease of synaptic transmission. Our findings suggest that FMRP is a regulator of synaptic vesicle dynamics, which supports the role of FMRP in presynaptic functions. Taken together, these studies provide novel insights into the molecular changes associated with FXS.

Identification and Characterization of lpfABCC′DE, a Fimbrial Operon of Enterohemorrhagic Escherichia coli O157:H7

PubMed Central

Torres, Alfredo G.; Giron, Jorge A.; Perna, Nicole T.; Burland, Valerie; Blattner, Fred R.; Avelino-Flores, Fabiola; Kaper, James B.

2002-01-01

The mechanisms underlying the adherence of Escherichia coli O157:H7 and other enterohemorrhagic E. coli (EHEC) strains to intestinal epithelial cells are poorly understood. We have identified a chromosomal region (designated lpfABCC′DE) in EHEC O157:H7 containing six putative open reading frames that was found to be closely related to the long polar (LP) fimbria operon (lpf) of Salmonella enterica serovar Typhimurium, both in gene order and in conservation of the deduced amino acid sequences. We show that lpfABCC′DE is organized as an operon and that its expression is induced during the exponential growth phase. The lpf genes from EHEC strain EDL933 were introduced into a nonfimbriated (Fim−) E. coli K-12 strain, and the transformed strain produced fimbriae as visualized by electron microscopy and adhered to tissue culture cells. Anti-LpfA antiserum recognized a ca. 16-kDa LpfA protein when expressed under regulation of the T7 promoter system. The antiserum also cross-reacted with the LP fimbriae in immunogold electron microscopy and Western blot experiments. Isogenic E. coli O157:H7 lpf mutants derived from strains 86-24 and AGT300 showed slight reductions in adherence to tissue culture cells and formed fewer microcolonies compared with their wild-type parent strains. The adherence and microcolony formation phenotypes were restored when the lpf operon was introduced on a plasmid. We propose that LP fimbriae participate in the interaction of E. coli O157:H7 with eukaryotic cells by assisting in microcolony formation. PMID:12228266

Comparative Transcriptomics of Bacillus mycoides Strains in Response to Potato-Root Exudates Reveals Different Genetic Adaptation of Endophytic and Soil Isolates.

PubMed

Yi, Yanglei; de Jong, Anne; Frenzel, Elrike; Kuipers, Oscar P

2017-01-01

Plant root secreted compounds alter the gene expression of associated microorganisms by acting as signal molecules that either stimulate or repel the interaction with beneficial or harmful species, respectively. However, it is still unclear whether two distinct groups of beneficial bacteria, non-plant-associated (soil) strains and plant-associated (endophytic) strains, respond uniformly or variably to the exposure with root exudates. Therefore, Bacillus mycoides , a potential biocontrol agent and plant growth-promoting bacterium, was isolated from the endosphere of potatoes and from soil of the same geographical region. Confocal fluorescence microscopy of plants inoculated with GFP-tagged B. mycoides strains showed that the endosphere isolate EC18 had a stronger plant colonization ability and competed more successfully for the colonization sites than the soil isolate SB8. To dissect these phenotypic differences, the genomes of the two strains were sequenced and the transcriptome response to potato root exudates was compared. The global transcriptome profiles evidenced that the endophytic isolate responded more pronounced than the soil-derived isolate and a higher number of significant differentially expressed genes were detected. Both isolates responded with the alteration of expression of an overlapping set of genes, which had previously been reported to be involved in plant-microbe interactions; including organic substance metabolism, oxidative reduction, and transmembrane transport. Notably, several genes were specifically upregulated in the endosphere isolate EC18, while being oppositely downregulated in the soil isolate SB8. These genes mainly encoded membrane proteins, transcriptional regulators or were involved in amino acid metabolism and biosynthesis. By contrast, several genes upregulated in the soil isolate SB8 and downregulated in the endosphere isolate EC18 were related to sugar transport, which might coincide with the different nutrient availability in the two environments. Altogether, the presented transcriptome profiles provide highly improved insights into the life strategies of plant-associated endophytes and soil isolates of B. mycoides .

Injectable nanosilica-chitosan microparticles for bone regeneration applications.

PubMed

Gaihre, Bipin; Lecka-Czernik, Beata; Jayasuriya, Ambalangodage C

2018-01-01

This study was aimed at assessing the effects of silica nanopowder incorporation into chitosan-tripolyphosphate microparticles with the ultimate goal of improving their osteogenic properties. The microparticles were prepared by simple coacervation technique and silica nanopowder was added at 0% (C), 2.5% (S1), 5% (S2) and 10% (S3) (w/w) to chitosan. We observed that this simple incorporation of silica nanopowder improved the growth and proliferation of osteoblasts along the surface of the microparticles. In addition, the composite microparticles also showed the increased expression of alkaline phosphatase and osteoblast specific genes. We observed a significant increase ( p < 0.05) in the expression of alkaline phosphatase by the cells growing on all sample groups compared to the control (C) groups at day 14. The morphological characterization of these microparticles through scanning electron microscopy showed that these microparticles were well suited to be used as the injectable scaffolds with perfectly spherical shape and size. The incorporation of silica nanopowder altered the nano-roughness of the microparticles as observed through atomic force microscopy scans with roughness values going down from C to S3. The results in this study, taken together, show the potential of chitosan-tripolyphosphate-silica nanopowder microparticles for improved bone regeneration applications.

The RootScope: a simple high-throughput screening system for quantitating gene expression dynamics in plant roots

PubMed Central

2013-01-01

Background High temperature stress responses are vital for plant survival. The mechanisms that plants use to sense high temperatures are only partially understood and involve multiple sensing and signaling pathways. Here we describe the development of the RootScope, an automated microscopy system for quantitating heat shock responses in plant roots. Results The promoter of Hsp17.6 was used to build a Hsp17.6p:GFP transcriptional reporter that is induced by heat shock in Arabidopsis. An automated fluorescence microscopy system which enables multiple roots to be imaged in rapid succession was used to quantitate Hsp17.6p:GFP response dynamics. Hsp17.6p:GFP signal increased with temperature increases from 28°C to 37°C. At 40°C the kinetics and localization of the response are markedly different from those at 37°C. This suggests that different mechanisms mediate heat shock responses above and below 37°C. Finally, we demonstrate that Hsp17.6p:GFP expression exhibits wave like dynamics in growing roots. Conclusions The RootScope system is a simple and powerful platform for investigating the heat shock response in plants. PMID:24119322

Anchoring of Heterochromatin to the Nuclear Lamina Reinforces Dosage Compensation-Mediated Gene Repression.

PubMed

Snyder, Martha J; Lau, Alyssa C; Brouhard, Elizabeth A; Davis, Michael B; Jiang, Jianhao; Sifuentes, Margarita H; Csankovszki, Györgyi

2016-09-01

Higher order chromosome structure and nuclear architecture can have profound effects on gene regulation. We analyzed how compartmentalizing the genome by tethering heterochromatic regions to the nuclear lamina affects dosage compensation in the nematode C. elegans. In this organism, the dosage compensation complex (DCC) binds both X chromosomes of hermaphrodites to repress transcription two-fold, thus balancing gene expression between XX hermaphrodites and XO males. X chromosome structure is disrupted by mutations in DCC subunits. Using X chromosome paint fluorescence microscopy, we found that X chromosome structure and subnuclear localization are also disrupted when the mechanisms that anchor heterochromatin to the nuclear lamina are defective. Strikingly, the heterochromatic left end of the X chromosome is less affected than the gene-rich middle region, which lacks heterochromatic anchors. These changes in X chromosome structure and subnuclear localization are accompanied by small, but significant levels of derepression of X-linked genes as measured by RNA-seq, without any observable defects in DCC localization and DCC-mediated changes in histone modifications. We propose a model in which heterochromatic tethers on the left arm of the X cooperate with the DCC to compact and peripherally relocate the X chromosomes, contributing to gene repression.

Anchoring of Heterochromatin to the Nuclear Lamina Reinforces Dosage Compensation-Mediated Gene Repression

PubMed Central

Brouhard, Elizabeth A.; Jiang, Jianhao; Sifuentes, Margarita H.

2016-01-01

Higher order chromosome structure and nuclear architecture can have profound effects on gene regulation. We analyzed how compartmentalizing the genome by tethering heterochromatic regions to the nuclear lamina affects dosage compensation in the nematode C. elegans. In this organism, the dosage compensation complex (DCC) binds both X chromosomes of hermaphrodites to repress transcription two-fold, thus balancing gene expression between XX hermaphrodites and XO males. X chromosome structure is disrupted by mutations in DCC subunits. Using X chromosome paint fluorescence microscopy, we found that X chromosome structure and subnuclear localization are also disrupted when the mechanisms that anchor heterochromatin to the nuclear lamina are defective. Strikingly, the heterochromatic left end of the X chromosome is less affected than the gene-rich middle region, which lacks heterochromatic anchors. These changes in X chromosome structure and subnuclear localization are accompanied by small, but significant levels of derepression of X-linked genes as measured by RNA-seq, without any observable defects in DCC localization and DCC-mediated changes in histone modifications. We propose a model in which heterochromatic tethers on the left arm of the X cooperate with the DCC to compact and peripherally relocate the X chromosomes, contributing to gene repression. PMID:27690361

Bm59 is an early gene, but is unessential for the propagation and assembly of Bombyx mori nucleopolyhedrovirus.

PubMed

Hu, Xiaolong; Shen, Yunwang; Zheng, Qin; Wang, Guobao; Wu, Xiaofeng; Gong, Chengliang

2016-02-01

Bombyx mori nucleopolyhedrovirus (BmNPV) is a major pathogen that specifically infects the domestic silkworm and causes serious economic loss to sericulture around the world. The function of BmNPV Bm59 gene in the viral life cycle is inconclusive. To investigate the role of Bm59 during viral infection, the transcription initiation site and temporal expression of Bm59 were analyzed, and Bm59-knockout virus was generated through homologous recombination in Escherichia coli. The results showed that Bm59 is an early transcription gene with an atypia early transcriptional start motif. Budded virion (BV) production and DNA replication in the BmN cells transfected with the Bm59-knockout virus bacmid were similar to those in the cells transfected with the wild-type virus. Electron microscopy revealed that the occlusion-derived virus can be produced in cells infected with the Bm59-knockout virus. These results indicated that Bm59 is an early gene and is not essential for viral replication or assembly of BmNPV. These findings suggested that non-essential gene (Bm59) remained in the viral genome, which may interact with other viral/host genes in a certain situation.

Quantitative measurement of intracellular protein dynamics using photobleaching or photoactivation of fluorescent proteins.

PubMed

Matsuda, Tomoki; Nagai, Takeharu

2014-12-01

Unlike in vitro protein dynamics, intracellular protein dynamics are intricately regulated by protein-protein interactions or interactions between proteins and other cellular components, including nucleic acids, the plasma membrane and the cytoskeleton. Alteration of these dynamics plays a crucial role in physiological phenomena such as gene expression and cell division. Live-cell imaging via microscopy with the inherent properties of fluorescent proteins, i.e. photobleaching and photoconversion, or fluorescence correlation spectroscopy, provides insight into the movement of proteins and their interactions with cellular components. This article reviews techniques based on photo-induced changes in the physicochemical properties of fluorescent proteins to measure protein dynamics inside living cells, and it also discusses the strengths and weaknesses of these techniques. © The Author 2014. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Magnetic Resonance Microscopy (MRM) of Single Mammalian Myofibers and Myonuclei.

PubMed

Lee, Choong H; Bengtsson, Niclas; Chrzanowski, Stephen M; Flint, Jeremy J; Walter, Glenn A; Blackband, Stephen J

2017-01-03

Recently, the first magnetic resonance microscopy (MRM) images at the cellular level in isolated mammalian brain tissues were obtained using microsurface coils. These methods can elucidate the cellular origins of MR signals and describe how these signals change over the course of disease progression and therapy. In this work, we explore the capability of these microimaging techniques to visualize mouse muscle fibers and their nuclei. Isolated myofibers expressing lacZ were imaged with and without a stain for β-galactosidase activity (S-Gal + ferric ammonium citrate) that produces both optical and MR contrast. We found that MRM can be used to image single myofibers with 6-μm resolution. The ability to image single myofibers will serve as a valuable tool to study MR properties attributed to healthy and myopathic cells. The ability to image nuclei tagged with MR/Optical gene markers may also find wide use in cell lineage MRI studies.

Magnetic Resonance Microscopy (MRM) of Single Mammalian Myofibers and Myonuclei

PubMed Central

Lee, Choong H.; Bengtsson, Niclas; Chrzanowski, Stephen M.; Flint, Jeremy J.; Walter, Glenn A.; Blackband, Stephen J.

2017-01-01

Recently, the first magnetic resonance microscopy (MRM) images at the cellular level in isolated mammalian brain tissues were obtained using microsurface coils. These methods can elucidate the cellular origins of MR signals and describe how these signals change over the course of disease progression and therapy. In this work, we explore the capability of these microimaging techniques to visualize mouse muscle fibers and their nuclei. Isolated myofibers expressing lacZ were imaged with and without a stain for β-galactosidase activity (S-Gal + ferric ammonium citrate) that produces both optical and MR contrast. We found that MRM can be used to image single myofibers with 6-μm resolution. The ability to image single myofibers will serve as a valuable tool to study MR properties attributed to healthy and myopathic cells. The ability to image nuclei tagged with MR/Optical gene markers may also find wide use in cell lineage MRI studies. PMID:28045071

3D Protein Dynamics in the Cell Nucleus.

PubMed

Singh, Anand P; Galland, Rémi; Finch-Edmondson, Megan L; Grenci, Gianluca; Sibarita, Jean-Baptiste; Studer, Vincent; Viasnoff, Virgile; Saunders, Timothy E

2017-01-10

The three-dimensional (3D) architecture of the cell nucleus plays an important role in protein dynamics and in regulating gene expression. However, protein dynamics within the 3D nucleus are poorly understood. Here, we present, to our knowledge, a novel combination of 1) single-objective based light-sheet microscopy, 2) photoconvertible proteins, and 3) fluorescence correlation microscopy, to quantitatively measure 3D protein dynamics in the nucleus. We are able to acquire >3400 autocorrelation functions at multiple spatial positions within a nucleus, without significant photobleaching, allowing us to make reliable estimates of diffusion dynamics. Using this tool, we demonstrate spatial heterogeneity in Polymerase II dynamics in live U2OS cells. Further, we provide detailed measurements of human-Yes-associated protein diffusion dynamics in a human gastric cancer epithelial cell line. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Proteomic profile of Mycobacterium tuberculosis after eupomatenoid-5 induction reveals potential drug targets.

PubMed

Ghiraldi-Lopes, Luciana D; Campanerut-Sá, Paula Az; Meneguello, Jean E; Seixas, Flávio Av; Lopes-Ortiz, Mariana A; Scodro, Regiane Bl; Pires, Claudia Ta; da Silva, Rosi Z; Siqueira, Vera Ld; Nakamura, Celso V; Cardoso, Rosilene F

2017-08-01

We investigated a proteome profile, protein-protein interaction and morphological changes of Mycobacterium tuberculosis after different times of eupomatenoid-5 (EUP-5) induction to evaluate the cellular response to the drug-induced damages. The bacillus was induced to sub-minimal inhibitory concentration of EUP-5 at 12 h, 24 h and 48 h. The proteins were separated by 2D gel electrophoresis, identified by LC/MS-MS. Scanning electron microscopy and Search Tool for the Retrieval of Interacting Genes/Proteins analyses were performed. EUP-5 impacts mainly in M. tuberculosis proteins of intermediary metabolism and interactome suggests a multisite disturbance that contributes to bacilli death. Scanning electron microscopy revealed the loss of bacillary form. Some of the differentially expressed proteins have the potential to be drug targets such as citrate synthase (Rv0896), phosphoglycerate kinase (Rv1437), ketol-acid reductoisomerase (Rv3001c) and ATP synthase alpha chain (Rv1308).

Identification of three wheat globulin genes by screening a Triticum aestivum BAC genomic library with cDNA from a diabetes-associated globulin

PubMed Central

Loit, Evelin; Melnyk, Charles W; MacFarlane, Amanda J; Scott, Fraser W; Altosaar, Illimar

2009-01-01

Background Exposure to dietary wheat proteins in genetically susceptible individuals has been associated with increased risk for the development of Type 1 diabetes (T1D). Recently, a wheat protein encoded by cDNA WP5212 has been shown to be antigenic in mice, rats and humans with autoimmune T1D. To investigate the genomic origin of the identified wheat protein cDNA, a hexaploid wheat genomic library from Glenlea cultivar was screened. Results Three unique wheat globulin genes, Glo-3A, Glo3-B and Glo-3C, were identified. We describe the genomic structure of these genes and their expression pattern in wheat seeds. The Glo-3A gene shared 99% identity with the cDNA of WP5212 at the nucleotide and deduced amino acid level, indicating that we have identified the gene(s) encoding wheat protein WP5212. Southern analysis revealed the presence of multiple copies of Glo-3-like sequences in all wheat samples, including hexaploid, tetraploid and diploid species wheat seed. Aleurone and embryo tissue specificity of WP5212 gene expression, suggested by promoter region analysis, which demonstrated an absence of endosperm specific cis elements, was confirmed by immunofluorescence microscopy using anti-WP5212 antibodies. Conclusion Taken together, the results indicate that a diverse group of globulins exists in wheat, some of which could be associated with the pathogenesis of T1D in some susceptible individuals. These data expand our knowledge of specific wheat globulins and will enable further elucidation of their role in wheat biology and human health. PMID:19615078

Thiol synthetases of legumes: immunogold localization and differential gene regulation by phytohormones

PubMed Central

Clemente, Maria R.; Bustos-Sanmamed, Pilar; Loscos, Jorge; James, Euan K.; Pérez-Rontomé, Carmen; Navascués, Joaquín; Gay, Marina; Becana, Manuel

2012-01-01

In plants and other organisms, glutathione (GSH) biosynthesis is catalysed sequentially by γ-glutamylcysteine synthetase (γECS) and glutathione synthetase (GSHS). In legumes, homoglutathione (hGSH) can replace GSH and is synthesized by γECS and a specific homoglutathione synthetase (hGSHS). The subcellular localization of the enzymes was examined by electron microscopy in several legumes and gene expression was analysed in Lotus japonicus plants treated for 1–48 h with 50 μM of hormones. Immunogold localization studies revealed that γECS is confined to chloroplasts and plastids, whereas hGSHS is also in the cytosol. Addition of hormones caused differential expression of thiol synthetases in roots. After 24–48 h, abscisic and salicylic acids downregulated GSHS whereas jasmonic acid upregulated it. Cytokinins and polyamines activated GSHS but not γECS or hGSHS. Jasmonic acid elicited a coordinated response of the three genes and auxin induced both hGSHS expression and activity. Results show that the thiol biosynthetic pathway is compartmentalized in legumes. Moreover, the similar response profiles of the GSH and hGSH contents in roots of non-nodulated and nodulated plants to the various hormonal treatments indicate that thiol homeostasis is independent of the nitrogen source of the plants. The differential regulation of the three mRNA levels, hGSHS activity, and thiol contents by hormones indicates a fine control of thiol biosynthesis at multiple levels and strongly suggests that GSH and hGSH play distinct roles in plant development and stress responses. PMID:22442424

Reactive oxygen species in the presence of high glucose alter ureteric bud morphogenesis.

PubMed

Zhang, Shao-Ling; Chen, Yun-Wen; Tran, Stella; Chenier, Isabelle; Hébert, Marie-Josée; Ingelfinger, Julie R

2007-07-01

Renal malformations are a major cause of childhood renal failure. During the development of the kidney, ureteric bud (UB) branching morphogenesis is critical for normal nephrogenesis. These studies investigated whether renal UB branching morphogenesis is altered by a high ambient glucose environment and studied underlying mechanism(s). Kidney explants that were isolated from different periods of gestation (embryonic days 12 to 18) from Hoxb7-green fluorescence protein mice were cultured for 24 h in either normal d-glucose (5 mM) or high d-glucose (25 mM) medium with or without various inhibitors. Alterations in renal morphogenesis were assessed by fluorescence microscopy. Paired-homeobox 2 (Pax-2) gene expression was determined by real-time quantitative PCR, Western blotting, and immunohistology. The results revealed that high d-glucose (25 mM) specifically stimulates UB branching morphogenesis via Pax-2 gene expression, whereas other glucose analogs, such as d-mannitol, l-glucose, and 2-deoxy-d-glucose, had no effect. The stimulatory effect of high glucose on UB branching was blocked in the presence of catalase and inhibitors of NADPH oxidase, mitochondrial electron transport chain complex I, and Akt signaling. Moreover, in in vivo studies, it seems that high glucose induces, via Pax-2 (mainly localized in UB), acceleration of UB branching but not nephron formation. Taken together, these data demonstrate that high glucose alters UB branching morphogenesis. This occurs, at least in part, via reactive oxygen species generation, activation of Akt signaling, and upregulation of Pax-2 gene expression.

Functional Analysis of the Chaperone-Usher Fimbrial Gene Clusters of Salmonella enterica serovar Typhi.

PubMed

Dufresne, Karine; Saulnier-Bellemare, Julie; Daigle, France

2018-01-01

The human-specific pathogen Salmonella enterica serovar Typhi causes typhoid, a major public health issue in developing countries. Several aspects of its pathogenesis are still poorly understood. S . Typhi possesses 14 fimbrial gene clusters including 12 chaperone-usher fimbriae ( stg, sth, bcf , fim, saf , sef , sta, stb, stc, std, ste , and tcf ). These fimbriae are weakly expressed in laboratory conditions and only a few are actually characterized. In this study, expression of all S . Typhi chaperone-usher fimbriae and their potential roles in pathogenesis such as interaction with host cells, motility, or biofilm formation were assessed. All S . Typhi fimbriae were better expressed in minimal broth. Each system was overexpressed and only the fimbrial gene clusters without pseudogenes demonstrated a putative major subunits of about 17 kDa on SDS-PAGE. Six of these (Fim, Saf, Sta, Stb, Std, and Tcf) also show extracellular structure by electron microscopy. The impact of fimbrial deletion in a wild-type strain or addition of each individual fimbrial system to an S . Typhi afimbrial strain were tested for interactions with host cells, biofilm formation and motility. Several fimbriae modified bacterial interactions with human cells (THP-1 and INT-407) and biofilm formation. However, only Fim fimbriae had a deleterious effect on motility when overexpressed. Overall, chaperone-usher fimbriae seem to be an important part of the balance between the different steps (motility, adhesion, host invasion and persistence) of S . Typhi pathogenesis.

Intestinal anti-inflammatory activity of Ground Cherry (Physalis angulata L.) standardized CO2 phytopharmaceutical preparation.

PubMed

Almeida Junior, Luiz Domingues; Quaglio, Ana Elisa Valencise; de Almeida Costa, Celso Acácio Rodrigues; Di Stasi, Luiz Claudio

2017-06-28

To investigate the effects of Ground Cherry ( Physalis angulata L.) standardized supercritical CO 2 extract in trinitrobenzenesulphonic acid (TNBS) model of rat intestinal inflammation. The animals were divided into groups that received vehicle or P. angulata extract (PACO 2 ) orally at the doses 25, 50 and 100 mg/kg daily by 5 d before TNBS damage. Protective effects of PACO 2 were assessed by macroscopic analysis, biochemical determinations of the levels of myeloperoxidase (MPO), alkaline phosphatase (ALP), glutathione and cytokines (such as INF-γ, IL-1β, IL-6, IL-10 and TNF-α), gene expression evaluation (including Hsp70, heparanase, NF-κB, mitogen-activated protein kinases (Mapk) 1, 3, 6 and 9, and the mucins genes Muc 1, 2, 3 and 4) and histopathological studies using optical, and electronic (transmission and scanning) microscopy. PACO 2 extract promoted a significant reduction in MPO and ALP activities, reducing oxidative stress and neutrophil infiltration. These effects were accompanied by significant reduction of colonic levels of IFN-γ and IL-6 and down-regulation of heparanase, Hsp70, Mapk3, Mapk9, Muc1 and Muc2 genes expression when compared with TNBS-control animals. In addition, protective effects were also evidenced by reduced neutrophil infiltration, recovery of cell architecture and replacement of mucin by histopathological and ultrastructural analysis. Physalis angulata supercritical CO 2 extract is an intestinal anti-inflammatory product that modulates oxidative stress, immune response and expression of inflammatory mediators, with potentially utility for treating inflammatory bowel disease.

Expression of the rabies virus glycoprotein in transgenic tomatoes.

PubMed

McGarvey, P B; Hammond, J; Dienelt, M M; Hooper, D C; Fu, Z F; Dietzschold, B; Koprowski, H; Michaels, F H

1995-12-01

We have engineered tomato plants (Lycopersicon esculentum Mill var. UC82b) to express a gene for the glycoprotein (G-protein), which coats the outer surface of the rabies virus. The recombinant constructs contained the G-protein gene from the ERA strain of rabies virus, including the signal peptide, under the control of the 35S promoter of cauliflower mosaic virus. Plants were transformed by Agrobacterium tumefaciens-mediated transformation of cotyledons and tissue culture on selective media. PCR confirmed the presence of the G-protein gene in plants surviving selection. Northern blot analysis indicated that RNA of the appropriate molecular weight was produced in both leaves and fruit of the transgenic plants. The recombinant G-protein was immunoprecipitated and detected by Western blot from leaves and fruit using different antisera. The G-protein expressed in tomato appeared as two distinct bands with apparent molecular mass of 62 and 60 kDa as compared to the 66 kDa observed for G-protein from virus grown in BHK cells. Electron microscopy of leaf tissue using immunogold-labeling and antisera specific for rabies G-protein showed localization of the G-protein to the Golgi bodies, vesicles, plasmalemma and cell walls of vascular parenchyma cells. In light of our previous demonstration that orally administered rabies G-protein from the same ERA strain elicits protective immunity in animals, these transgenic plants should provide a valuable tool for the development of edible oral vaccines.

Optimal 3D culture of primary articular chondrocytes for use in the rotating wall vessel bioreactor.

PubMed

Mellor, Liliana F; Baker, Travis L; Brown, Raquel J; Catlin, Lindsey W; Oxford, Julia Thom

2014-08-01

Reliable culturing methods for primary articular chondrocytes are essential to study the effects of loading and unloading on joint tissue at the cellular level. Due to the limited proliferation capacity of primary chondrocytes and their tendency to dedifferentiate in conventional culture conditions, long-term culturing conditions of primary chondrocytes can be challenging. The goal of this study was to develop a suspension culturing technique that not only would retain the cellular morphology, but also maintain the gene expression characteristics of primary articular chondrocytes. Three-dimensional culturing methods were compared and optimized for primary articular chondrocytes in the rotating wall vessel bioreactor, which changes the mechanical culture conditions to provide a form of suspension culture optimized for low shear and turbulence. We performed gene expression analysis and morphological characterization of cells cultured in alginate beads, Cytopore-2 microcarriers, primary monolayer culture, and passaged monolayer cultures using reverse transcription-PCR and laser scanning confocal microscopy. Primary chondrocytes grown on Cytopore-2 microcarriers maintained the phenotypical morphology and gene expression pattern observed in primary bovine articular chondrocytes, and retained these characteristics for up to 9 d. Our results provide a novel and alternative culturing technique for primary chondrocytes suitable for studies that require suspension such as those using the rotating wall vessel bioreactor. In addition, we provide an alternative culturing technique for primary chondrocytes that can impact future mechanistic studies of osteoarthritis progression, treatments for cartilage damage and repair, and cartilage tissue engineering.

Thiol synthetases of legumes: immunogold localization and differential gene regulation by phytohormones.

PubMed

Clemente, Maria R; Bustos-Sanmamed, Pilar; Loscos, Jorge; James, Euan K; Pérez-Rontomé, Carmen; Navascués, Joaquín; Gay, Marina; Becana, Manuel

2012-06-01

In plants and other organisms, glutathione (GSH) biosynthesis is catalysed sequentially by γ-glutamylcysteine synthetase (γECS) and glutathione synthetase (GSHS). In legumes, homoglutathione (hGSH) can replace GSH and is synthesized by γECS and a specific homoglutathione synthetase (hGSHS). The subcellular localization of the enzymes was examined by electron microscopy in several legumes and gene expression was analysed in Lotus japonicus plants treated for 1-48 h with 50 μM of hormones. Immunogold localization studies revealed that γECS is confined to chloroplasts and plastids, whereas hGSHS is also in the cytosol. Addition of hormones caused differential expression of thiol synthetases in roots. After 24-48 h, abscisic and salicylic acids downregulated GSHS whereas jasmonic acid upregulated it. Cytokinins and polyamines activated GSHS but not γECS or hGSHS. Jasmonic acid elicited a coordinated response of the three genes and auxin induced both hGSHS expression and activity. Results show that the thiol biosynthetic pathway is compartmentalized in legumes. Moreover, the similar response profiles of the GSH and hGSH contents in roots of non-nodulated and nodulated plants to the various hormonal treatments indicate that thiol homeostasis is independent of the nitrogen source of the plants. The differential regulation of the three mRNA levels, hGSHS activity, and thiol contents by hormones indicates a fine control of thiol biosynthesis at multiple levels and strongly suggests that GSH and hGSH play distinct roles in plant development and stress responses.

Mechanism of biological denitrification inhibition: procyanidins induce an allosteric transition of the membrane-bound nitrate reductase through membrane alteration.

PubMed

Bardon, Clément; Poly, Franck; Piola, Florence; Pancton, Muriel; Comte, Gilles; Meiffren, Guillaume; Haichar, Feth el Zahar

2016-05-01

Recently, it has been shown that procyanidins from Fallopia spp. inhibit bacterial denitrification, a phenomenon called biological denitrification inhibition (BDI). However, the mechanisms involved in such a process remain unknown. Here, we investigate the mechanisms of BDI involving procyanidins, using the model strain Pseudomonas brassicacearum NFM 421. The aerobic and anaerobic (denitrification) respiration, cell permeability and cell viability of P. brassicacearum were determined as a function of procyanidin concentration. The effect of procyanidins on the bacterial membrane was observed using transmission electronic microscopy. Bacterial growth, denitrification, NO3- and NO2-reductase activity, and the expression of subunits of NO3- (encoded by the gene narG) and NO2-reductase (encoded by the gene nirS) under NO3 or NO2 were measured with and without procyanidins. Procyanidins inhibited the denitrification process without affecting aerobic respiration at low concentrations. Procyanidins also disturbed cell membranes without affecting cell viability. They specifically inhibited NO3- but not NO2-reductase.Pseudomonas brassicacearum responded to procyanidins by over-expression of the membrane-bound NO3-reductase subunit (encoded by the gene narG). Our results suggest that procyanidins can specifically inhibit membrane-bound NO3-reductase inducing enzymatic conformational changes through membrane disturbance and that P. brassicacearum responds by over-expressing membrane-bound NO3-reductase. Our results lead the way to a better understanding of BDI. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Upregulation of mucin glycoprotein MUC1 in the progression to esophageal adenocarcinoma and therapeutic potential with a targeted photoactive antibody-drug conjugate

PubMed Central

Haidry, Rehan J.; Oukrif, Dahmane; Khan, Saif-U-Rehman; Puccio, Ignazio; Gandy, Michael; Reinert, Halla W.; Bloom, Ellie; Rashid, Mohammed; Yahioglu, Gokhan; Deonarain, Mahendra P.; Hamoudi, Rifat; Rodriguez-Justo, Manuel; Novelli, Marco R.; Lovat, Laurence B.

2017-01-01

Background Mucin glycoprotein 1 (MUC1) is a glycosylated transmembrane protein on epithelial cells. We investigate MUC1 as a therapeutic target in Barrett’s epithelium (BE) and esophageal adenocarcinoma (EA) and provide proof of concept for a light based therapy targeting MUC1. RESULTS MUC1 was present in 21% and 30% of significantly enriched pathways comparing BE and EA to squamous epithelium respectively. MUC1 gene expression was x2.3 and x2.2 higher in BE (p=<0.001) and EA (p=0.03). MUC1 immunohistochemical expression increased during progression to EA and followed tumor invasion. HuHMFG1 based photosensitive antibody drug conjugates (ADC) showed cell internalization, MUC1 selective and light-dependent cytotoxicity (p=0.0006) and superior toxicity over photosensitizer alone (p=0.0022). Methods Gene set enrichment analysis (GSEA) evaluated pathways during BE and EA development and quantified MUC1 gene expression. Immunohistochemistry and flow cytometry evaluated the anti-MUC1 antibody HuHMFG1 in esophageal cells of varying pathological grade. Confocal microscopy examined HuHMFG1 internalization and HuHMFG1 ADCs were created to deliver a MUC1 targeted phototoxic payload. Conclusions MUC1 is a promising target in EA. Molecular and light based targeting of MUC1 with a photosensitive ADC is effective in vitro and after development may enable treatment of locoregional tumors endoscopically. PMID:28212575

The broccoli (Brassica oleracea) phloem tissue proteome.

PubMed

Anstead, James A; Hartson, Steven D; Thompson, Gary A

2013-11-07

The transport of sugars, hormones, amino acids, proteins, sugar alcohols, and other organic compounds from the sites of synthesis to the sites of use or storage occurs through the conducting cells of the phloem. To better understand these processes a comprehensive understanding of the proteins involved is required. While a considerable amount of data has been obtained from proteomic analyses of phloem sap, this has mainly served to identify the soluble proteins that are translocated through the phloem network. In order to obtain more comprehensive proteomic data from phloem tissue we developed a simple dissection procedure to isolate phloem tissue from Brassica oleracea. The presence of a high density of phloem sieve elements was confirmed using light microscopy and fluorescently labeled sieve element-specific antibodies. To increase the depth of the proteomic analysis for membrane bound and associated proteins, soluble proteins were extracted first and subsequent extractions were carried out using two different detergents (SDS and CHAPSO). Across all three extractions almost four hundred proteins were identified and each extraction method added to the analysis demonstrating the utility of an approach combining several extraction protocols. The phloem was found to be enriched in proteins associated with biotic and abiotic stress responses and structural proteins. Subsequent expression analysis identified a number of genes that appear to be expressed exclusively or at very high levels in phloem tissue, including genes that are known to express specifically in the phloem as well as novel phloem genes.

Black Raspberry-Derived Anthocyanins Demethylate Tumor Suppressor Genes Through the Inhibition of DNMT1 and DNMT3B in Colon Cancer Cells

PubMed Central

Wang, Li-Shu; Kuo, Chieh-Ti; Cho, Seung-Ju; Seguin, Claire; Siddiqui, Jibran; Stoner, Kristen; Weng, Yu-I; Huang, Tim H.-M.; Tichelaar, Jay; Yearsley, Martha; Stoner, Gary D.; Huang, Yi-Wen

2013-01-01

We previously reported that oral administration of black raspberry powder decreased promoter methylation of tumor suppressor genes in tumors from patients with colorectal cancer. The anthocyanins (ACs) in black raspberries are responsible, at least in part, for their cancer-inhibitory effects. In the present study, we asked if ACs are responsible for the demethylation effects observed in colorectal cancers. Three days of treatment of ACs at 0.5, 5, and 25 μg/ml suppressed activity and protein expression of DNMT1 and DNMT3B in HCT116, Caco2 and SW480 cells. Promoters of CDKN2A, and SFRP2, SFRP5, and WIF1, upstream of Wnt pathway, were demethylated by ACs. mRNA expression of some of these genes was increased. mRNA expression of β-catenin and c-Myc, downstream of Wnt pathway, and cell proliferation were decreased; apoptosis was increased. ACs were taken up into HCT116 cells and were differentially localized with DNMT1 and DNMT3B in the same cells visualized using confocal laser scanning microscopy. Although it was reported that DNMT3B is regulated by c-Myc in mouse lymphoma, DNMT3B did not bind with c-Myc in HCT116 cells. In conclusion, our results suggest that ACs are responsible, at least in part, for the demethylation effects of whole black raspberries in colorectal cancers. PMID:23368921

F463L increases the potential of dofetilide on human ether-a-go-go-related gene (hERG) channels.

PubMed

Cheng, Gong; Wu, Jine; Han, Wenqi; Sun, Chaofeng

2018-06-01

Mutations in genes related to long QT syndrome (LQTS) is recognized as an independent risk of drug-induced LQTS. We previously screened a mutation F463L in a Chinese patient with LQT2, syncope, and epilepsy. Here, we planned to illustrate how F463L influences the action of dofetilide on hERG channels. F463L-hERG plasmids were transfected into the stable Human Embryonic Kidney 293 (HEK293) cells expressing WT-hERG to generate heterozygous mutant (WT + F463L-hERG). Whole-cell patch clamp and laser confocal scanning microscopy were used to evaluate electrophysiological consequences and the membrane distribution of hERG protein. In comparison of WT-hERG channels exposed to dofetilide, heterozygous F463L-hERG channels showed a reduction in the density of tail currents when exposed amidarone. F463L-hERG also altered the action of dofetilide on the gating properties of hERG channels. Images of dofetilide-treated cells expressing heterozygous F463L showed a severe retention and reduction of protein expression on the membrane compared to WT. In conclusion, dofetilide displays a powerful inhibitory effect on the currents from cells expressing heterozygous F463L, thus showing an additive suppression of currents by F463L with dofetilide. © 2018 Wiley Periodicals, Inc.

Protease Expression Levels in Prostate Cancer Tissue Can Explain Prostate Cancer-Associated Seminal Biomarkers-An Explorative Concept Study.

PubMed

Neuhaus, Jochen; Schiffer, Eric; Mannello, Ferdinando; Horn, Lars-Christian; Ganzer, Roman; Stolzenburg, Jens-Uwe

2017-05-04

Previously, we described prostate cancer (PCa) detection (83% sensitivity; 67% specificity) in seminal plasma by CE-MS/MS. Moreover, advanced disease was distinguished from organ-confined tumors with 80% sensitivity and 82% specificity. The discovered biomarkers were naturally occurring fragments of larger seminal proteins, predominantly semenogelin 1 and 2, representing endpoints of the ejaculate liquefaction. Here we identified proteases putatively involved in PCa specific protein cleavage, and examined gene expression and tissue protein levels, jointly with cell localization in normal prostate (nP), benign prostate hyperplasia (BPH), seminal vesicles and PCa using qPCR, Western blotting and confocal laser scanning microscopy. We found differential gene expression of chymase (CMA1), matrix metalloproteinases (MMP3, MMP7), and upregulation of MMP14 and tissue inhibitors (TIMP1 and TIMP2) in BPH. In contrast tissue protein levels of MMP14 were downregulated in PCa. MMP3/TIMP1 and MMP7/TIMP1 ratios were decreased in BPH. In seminal vesicles, we found low-level expression of most proteases and, interestingly, we also detected TIMP1 and low levels of TIMP2. We conclude that MMP3 and MMP7 activity is different in PCa compared to BPH due to fine regulation by their inhibitor TIMP1. Our findings support the concept of seminal plasma biomarkers as non-invasive tool for PCa detection and risk stratification.

The use of quaternised chitosan-loaded PMMA to inhibit biofilm formation and downregulate the virulence-associated gene expression of antibiotic-resistant staphylococcus.

PubMed

Tan, Honglue; Peng, Zhaoxiang; Li, Qingtian; Xu, Xiaofen; Guo, Shengrong; Tang, Tingting

2012-01-01

Biomaterial-associated infections remain a serious complication in orthopaedic surgery. Treatments, including the local use of antibiotic-loaded polymethylmethacrylate (PMMA) bone cement, are not always successful because of multiantibiotic-resistant organisms. In this study, we synthesised a new quaternised chitosan derivative (hydroxypropyltrimethyl ammonium chloride chitosan, HACC) that contains a series of substitutions of quaternary ammonium and demonstrated that HACC with a 26% degree of substitution (DS; referred to as 26%HACC) had a strong antibacterial activity and simultaneously good biocompatibility with osteogenic cells. We loaded 26%HACC at 20% by weight into PMMA bone cement to investigate whether HACC in PMMA prevents bacterial biofilm formation on the surface of bone cements. Chitosan-loaded PMMA (at the same weight ratio), gentamicin-loaded PMMA and PMMA with no antibiotic were also investigated and compared. Two clinical isolates, Staphylococcus epidermidis 389 and methicillin-resistant S. epidermidis (MRSE287), and two standard strains, S. epidermidis (ATCC35984) and methicillin-resistant Staphylococcus aureus (ATCC43300), were selected to evaluate the bacterial biofilm formation at 6, 12 and 24 h using the spread plate method, confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). The results showed that 26%HACC-loaded PMMA inhibited biofilm formation on its surface, while the PMMA control and chitosan-loaded PMMA were unable to inhibit biofilm formation. The gentamicin-loaded PMMA decreased the number of viable methicillin-resistant Staphylococcus strains, but its ability to inhibit biofilm formation was lower than 26%HACC-loaded PMMA. Real-time PCR demonstrated that 26%HACC-loaded PMMA markedly downregulated the expression of icaAD, which encodes essential enzymes for polysaccharide intercellular adhesion (PIA) biosynthesis, upregulated the expression level of icaR, which negatively mediates icaAD expression, and also downregulated the expression of MecA, which encodes membrane-bound enzymes known to be penicillin-binding proteins. Our study indicates that 26%HACC-loaded PMMA prevents biofilm formation of Staphylococcus, including antibiotic-resistant strains, on the surface of bone cement, and downregulates the virulence-associated gene expression of antibiotic-resistant staphylococcus, thus providing a promising new strategy for combating implant infections and osteomyelitis. Copyright © 2011 Elsevier Ltd. All rights reserved.

Cytoskeletal dynamics in interphase, mitosis and cytokinesis analysed through Agrobacterium-mediated transient transformation of tobacco BY-2 cells.

PubMed

Buschmann, H; Green, P; Sambade, A; Doonan, J H; Lloyd, C W

2011-04-01

Transient transformation with Agrobacterium is a widespread tool allowing rapid expression analyses in plants. However, the available methods generate expression in interphase and do not allow the routine analysis of dividing cells. Here, we present a transient transformation method (termed 'TAMBY2') to enable cell biological studies in interphase and cell division. Agrobacterium-mediated transient gene expression in tobacco BY-2 was analysed by Western blotting and quantitative fluorescence microscopy. Time-lapse microscopy of cytoskeletal markers was employed to monitor cell division. Double-labelling in interphase and mitosis enabled localization studies. We found that the transient transformation efficiency was highest when BY-2/Agrobacterium co-cultivation was performed on solid medium. Transformants produced in this way divided at high frequency. We demonstrated the utility of the method by defining the behaviour of a previously uncharacterized microtubule motor, KinG, throughout the cell cycle. Our analyses demonstrated that TAMBY2 provides a flexible tool for the transient transformation of BY-2 with Agrobacterium. Fluorescence double-labelling showed that KinG localizes to microtubules and to F-actin. In interphase, KinG accumulates on microtubule lagging ends, suggesting a minus-end-directed function in vivo. Time-lapse studies of cell division showed that GFP-KinG strongly labels preprophase band and phragmoplast, but not the metaphase spindle. © 2010 The Authors. New Phytologist © 2010 New Phytologist Trust.

A rice chloroplast transit peptide sequence does not alter the cytoplasmic localization of sheep serotonin N-acetyltransferase expressed in transgenic rice plants.

PubMed

Byeon, Yeong; Lee, Hyoung Yool; Lee, Kyungjin; Back, Kyoungwhan

2014-09-01

Ectopic overexpression of melatonin biosynthetic genes of animal origin has been used to generate melatonin-rich transgenic plants to examine the functional roles of melatonin in plants. However, the subcellular localization of these proteins expressed in the transgenic plants remains unknown. We studied the localization of sheep (Ovis aries) serotonin N-acetyltransferase (OaSNAT) and a translational fusion of a rice SNAT transit peptide to OaSNAT (TS:OaSNAT) in plants. Laser confocal microscopy analysis revealed that both OaSNAT and TS:OaSNAT proteins were localized to the cytoplasm even with the addition of the transit sequence to OaSNAT. Transgenic rice plants overexpressing the TS:OaSNAT fusion transgene exhibited high SNAT enzyme activity relative to untransformed wild-type plants, but lower activity than transgenic rice plants expressing the wild-type OaSNAT gene. Melatonin levels in both types of transgenic rice plant corresponded well with SNAT enzyme activity levels. The TS:OaSNAT transgenic lines exhibited increased seminal root growth relative to wild-type plants, but less than in the OaSNAT transgenic lines, confirming that melatonin promotes root growth. Seed-specific OaSNAT expression under the control of a rice prolamin promoter did not confer high levels of melatonin production in transgenic rice seeds compared with seeds from transgenic plants expressing OaSNAT under the control of the constitutive maize ubiquitin promoter. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Zebrafish collagen XII is present in embryonic connective tissue sheaths (fascia) and basement membranes.

PubMed

Bader, Hannah L; Keene, Douglas R; Charvet, Benjamin; Veit, Guido; Driever, Wolfgang; Koch, Manuel; Ruggiero, Florence

2009-01-01

Connective tissues ensure the cohesion of the tissues of the body, but also form specialized structures such as tendon and bone. Collagen XII may enhance the stability of connective tissues by bridging collagen fibrils, but its function is still unclear. Here, we used the zebrafish model to visualize its expression pattern in the whole organism. The zebrafish col12a1 gene is homologous to the small isoform of the tetrapod col12a1 gene. In agreement with the biochemical data reported for the small isoform, the zebrafish collagen XII alpha1 chain was characterized as a collagenase sensitive band migrating at approximately 200 kDa. Using newly generated polyclonal antibodies and anti-sense probes, we performed a comprehensive analysis of its expression in developing zebrafish. Collagen XII exhibited a much broader expression pattern than previously thought: it was ubiquitously expressed in the connective tissue sheaths (fascia) that encase the tissues and organs of the body. For example, it was found in sclera, meninges, epimysia and horizontal and vertical myosepta. Collagen XII was also detected in head mesenchyme, pharyngeal arches and within the spinal cord, where it was first expressed within and then at the lateral borders of the floor plate and at the dorsal midline. Furthermore, double immunofluorescence staining with laminin and immunogold electron microscopy revealed that collagen XII is associated with basement membranes. These data suggest that collagen XII is implicated in tissue cohesion by stabilizing fascia and by linking fascia to basement membranes.

The LMNA mutation p.Arg321Ter associated with dilated cardiomyopathy leads to reduced expression and a skewed ratio of lamin A and lamin C proteins

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

Al-Saaidi, Rasha; Rasmussen, Torsten B.; Palmfeldt, Johan

2013-11-15

Dilated cardiomyopathy (DCM) is a disease of the heart muscle characterized by cardiac chamber enlargement and reduced systolic function of the left ventricle. Mutations in the LMNA gene represent the most frequent known genetic cause of DCM associated with disease of the conduction systems. The LMNA gene generates two major transcripts encoding the nuclear lamina major components lamin A and lamin C by alternative splicing. Both haploinsuffiency and dominant negative effects have been proposed as disease mechanism for premature termination codon (PTC) mutations in LMNA. These mechanisms however are still not clearly established. In this study, we used a representativemore » LMNA nonsense mutation, p.Arg321Ter, to shed light on the molecular disease mechanisms. Cultured fibroblasts from three DCM patients carrying this mutation were analyzed. Quantitative reverse transcriptase PCR and sequencing of these PCR products indicated that transcripts from the mutant allele were degraded by the nonsense-mediated mRNA decay (NMD) mechanism. The fact that no truncated mutant protein was detectable in western blot (WB) analysis strengthens the notion that the mutant transcript is efficiently degraded. Furthermore, WB analysis showed that the expression of lamin C protein was reduced by the expected approximately 50%. Clearly decreased lamin A and lamin C levels were also observed by immunofluorescence microscopy analysis. However, results from both WB and nano-liquid chromatography/mass spectrometry demonstrated that the levels of lamin A protein were more reduced suggesting an effect on expression of lamin A from the wild type allele. PCR analysis of the ratio of lamin A to lamin C transcripts showed unchanged relative amounts of lamin A transcript suggesting that the effect on the wild type allele was operative at the protein level. Immunofluorescence microscopy analysis showed no abnormal nuclear morphology of patient fibroblast cells. Based on these data, we propose that heterozygosity for the nonsense mutation causes NMD degradation of the mutant transcripts blocking expression of the truncated mutant protein and an additional trans effect on lamin A protein levels expressed from the wild type allele. We discuss the possibility that skewing of the lamin A to lamin C ratio may contribute to ensuing processes that destabilize cardiomyocytes and trigger cardiomyopathy - Highlights: • We study disease mechanisms in DCM patients carrying PTC mutations in the LMNA gene. • The mutant transcript is degraded by the nonsense mediated mRNA decay system. • Skewed lamin A to lamin C protein ratio expressed from the wild type allele. • We suggest a combined pathomechanism: haploinsuffiency plus lamin A/C imbalance.« less

Use of the yeast-like cells of Tremella fuciformis as a cell factory to produce a Pleurotus ostreatus hydrophobin.

PubMed

Zhu, Hanyu; Liu, Dongmei; Wang, Yuanyuan; Ren, Danfeng; Zheng, Liesheng; Chen, Liguo; Ma, Aimin

2017-08-01

To obtain hydrophobin, a Class I hydrophobin gene, Po.hyd from Pleurotus ostreatus, was transformed into the yeast-like cells of Tremella fuciformis using Agrobacterium tumefaciens. The hydrophobin Po.HYD from P. ostreatus was heterogeneously expressed by the yeast-like cells of T. fuciformis. Plasmids harboring the Po.hyd gene driven by endogenous glyceraldehyde-3-phosphate dehydrogenase promoter were transformed by A. tumefaciens. The integration and expression of the rPo.HYD in the T. fuciformis cells were confirmed by PCR, Southern blot, fluorescence microscopy and quantitative real-time PCR. SDS-PAGE demonstrated that the rPo.HYD was extracted with the expected MW of 14 kDa. The yield of purified rPo.HYD was 0.58 mg/g dry wt. The protein, with its ability to stabilize oil droplets, exhibited a better emulsifying activity than the typical food emulsifiers Tween 20 and sodium caseinate. Tremella fuciformis can be used as a cell factory to produce hydrophobin on a large scale for the food industry.

Epiphytic cyanobacteria of the seagrass Cymodocea rotundata: diversity, diel nifH expression and nitrogenase activity.

PubMed

Hamisi, Mariam; Díez, Beatriz; Lyimo, Thomas; Ininbergs, Karolina; Bergman, Birgitta

2013-06-01

Seagrasses are photoautotrophic, ecologically important components of many globally widespread coastal ecosystems, in which combined nitrogen may limit their production. We examined the biodiversity and diazotrophic capacity of microbial epiphytes associated with the phyllosphere of the seagrass Cymodocea rotundata of the Western Indian Ocean. Light microscopy, 16S rRNA and nifH gene analysis revealed the dominance of cyanobacteria in the epiphytic microbial community. Most phylotypes were related to free-living uncultured benthic cyanobacteria, while some to cyanobacterial endosymbionts of marine diatoms. Novel and potentially diazotrophic species, some of known pantropical distribution, were also discovered. Significant diel nitrogenase activities (acetylene reduction assay) were recorded (up to 358 ± 232 nmol C2H4 g(-1) of seagrass FW h(-1)). The nifH gene expression patterns showed that heterocystous phylotypes may be the dominant diazotrophs during the day and non-heterocystous at night. These data show that C. rotundata is colonized by diverse diazotrophic cyanobacteria species and suggest that these may be beneficial partners of seagrasses in nitrogen-depleted waters. © 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.

Inactivation of a putative efflux pump (LmrB) in Streptococcus mutans results in altered biofilm structure and increased exopolysaccharide synthesis: implications for biofilm resistance.

PubMed

Liu, Jia; Zhang, Jianying; Guo, Lihong; Zhao, Wei; Hu, Xiaoli; Wei, Xi

2017-07-01

Efflux pumps are a mechanism associated with biofilm formation and resistance. There is limited information regarding efflux pumps in Streptococcus mutans, a major pathogen in dental caries. The aim of this study was to investigate potential roles of a putative efflux pump (LmrB) in S. mutans biofilm formation and susceptibility. Upon lmrB inactivation and antimicrobial exposure, the biofilm structure and expression of other efflux pumps were examined using confocal laser scanning microscopy (CLSM) and qRT-PCR. lmrB inactivation resulted in biofilm structural changes, increased EPS formation and EPS-related gene transcription (p < 0.05), but no improvement in susceptibility was observed. The expression of most efflux pump genes increased upon lmrB inactivation when exposed to antimicrobials (p < 0.05), suggesting a feedback mechanism that activated the transcription of other efflux pumps to compensate for the loss of lmrB. These observations imply that sole inactivation of lmrB is not an effective solution to control biofilms.

Integrated time-lapse and single-cell transcription studies highlight the variable and dynamic nature of human hematopoietic cell fate commitment

PubMed Central

Moussy, Alice; Cosette, Jérémie; Parmentier, Romuald; da Silva, Cindy; Corre, Guillaume; Richard, Angélique; Gandrillon, Olivier; Stockholm, Daniel

2017-01-01

Individual cells take lineage commitment decisions in a way that is not necessarily uniform. We address this issue by characterising transcriptional changes in cord blood-derived CD34+ cells at the single-cell level and integrating data with cell division history and morphological changes determined by time-lapse microscopy. We show that major transcriptional changes leading to a multilineage-primed gene expression state occur very rapidly during the first cell cycle. One of the 2 stable lineage-primed patterns emerges gradually in each cell with variable timing. Some cells reach a stable morphology and molecular phenotype by the end of the first cell cycle and transmit it clonally. Others fluctuate between the 2 phenotypes over several cell cycles. Our analysis highlights the dynamic nature and variable timing of cell fate commitment in hematopoietic cells, links the gene expression pattern to cell morphology, and identifies a new category of cells with fluctuating phenotypic characteristics, demonstrating the complexity of the fate decision process (which is different from a simple binary switch between 2 options, as it is usually envisioned). PMID:28749943

Biocompatibility evaluation of HDPE-UHMWPE reinforced β-TCP nanocomposites using highly purified human osteoblast cells.

PubMed

Shokrgozar, M A; Farokhi, M; Rajaei, F; Bagheri, M H A; Azari, Sh; Ghasemi, I; Mottaghitalab, F; Azadmanesh, K; Radfar, J

2010-12-15

Biocompatibility of β-TCP/HDPE-UHMWPE nanocomposite as a new bone substitute material was evaluated by using highly purified human osteoblast cells. Human osteoblast cells were isolated from bone tissue and characterized by immunofluorescence Staining before and after purification using magnetic bead system. Moreover, proliferation, alkaline phosphatase production, cell attachment, calcium deposition, gene expression, and morphology of osteoblast cells on β-TCP/HDPE-UHMWPE nanocomposites were evaluated. The results have shown that the human osteoblast cells were successfully purified and were suitable for subsequent cell culturing process. The high proliferation rate of osteoblast cells on β-TCP/HDPE-UHMWPE nanocomposite confirmed the great biocompatibility of the scaffold. Expression of bone-specific genes was taken place after the cells were incubated in composite extract solutions. Furthermore, osteoblast cells were able to mineralize the matrix next to composite samples. Scanning electron microscopy demonstrated that cells had normal morphology on the scaffold. Thus, these results indicated that the nanosized β-TCP/HDPE-UHMWPE blend composites could be potential scaffold, which is used in bone tissue engineering. Copyright © 2010 Wiley Periodicals, Inc.