scholarly journals Identification of RISC-Associated Adenoviral MicroRNAs, a Subset of Their Direct Targets, and Global Changes in the Targetome upon Lytic Adenovirus 5 Infection

2014 ◽  
Vol 89 (3) ◽  
pp. 1608-1627 ◽  
Author(s):  
Florian Bellutti ◽  
Maximilian Kauer ◽  
Doris Kneidinger ◽  
Thomas Lion ◽  
Reinhard Klein
Keyword(s):  
Gene Expression ◽  
Relative Abundance ◽  
A549 Cells ◽  
Global Changes ◽  
Cellular Mirnas ◽  
Total Rna ◽  
Cellular Mrnas ◽  
Comprehensive Picture ◽  
Infected Cells ◽  
Adenovirus 5

ABSTRACTAdenoviruses encode a set of highly abundant microRNAs (mivaRNAs), which are generated by Dicer-mediated cleavage of the larger noncoding virus-associated RNAs (VA RNAs) I and II. We performed deep RNA sequencing to thoroughly investigate the relative abundance of individual single strands of mivaRNA isoforms in human A549 cells lytically infected with human adenovirus 5 (Ad5) at physiologically relevant multiplicities of infection (MOIs). In addition, we investigated their relative abundance in the endogenous RNA-induced silencing complexes (RISCs). The occupation of endogenous RISCs by mivaRNAs turned out to be pronounced but not as dominant as previously inferred from experiments with AGO2-overexpressing cells infected at high MOIs. In parallel, levels of RISC-incorporated mRNAs were investigated as well. Analysis of mRNAs enriched in RISCs in Ad5-infected cells revealed that only mRNAs with complementarity to the seed sequences of mivaRNAs derived from VA RNAI but not VA RNAII were overrepresented among them, indicating that only mivaRNAs derived from VA RNAI are likely to contribute substantially to the posttranscriptional downregulation of host gene expression. Furthermore, to generate a comprehensive picture of the entire transcriptome/targetome in lytically infected cells, we determined changes in cellular miRNA levels in both total RNA and RISC RNA as well, and bioinformatical analysis of mRNAs of total RNA/RISC fractions revealed a general, genome-wide trend toward detargeting of cellular mRNAs upon infection. Lastly, we identified the direct targets of both single strands of a VA RNAI-derived mivaRNA that constituted one of the two most abundant isoforms in RISCs of lytically infected A549 cells.IMPORTANCEViral and cellular miRNAs have been recognized as important players in virus-host interactions. This work provides the currently most comprehensive picture of the entire mRNA/miRNA transcriptome and of the complete RISC targetome during lytic adenovirus infection and thus represents the basis for a deeper understanding of the interplay between the virus and the cellular RNA interference machinery. Our data suggest that, at least in the model system that was employed, lytic infection by Ad5 is accompanied by a measurable global net detargeting effect on cellular mRNAs, and analysis of RISC-associated viral small RNAs revealed that the VA RNAs are the only source of virus-encoded miRNAs. Moreover, this work allows to assess the power of individual viral miRNAs to regulate cellular gene expression and provides a list of proven and putative direct targets of these miRNAs, which is of importance, given the fact that information about validated targets of adenovirus-encoded miRNAs is scarce.

198 PROGESTERONE, ESTROGEN (ER-α AND ER-β), AND OXYTOCIN RECEPTOR GENE EXPRESSION IN CANINE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 248
Author(s):  
A. A. P. Derussi ◽  
A. C. S. Castilho ◽  
R. W. A. Souza ◽  
R. Volpato ◽  
C. R. F. Guaitolini ◽  
...  
Keyword(s):  
Gene Expression ◽  
Relative Abundance ◽  
Hormone Receptors ◽  
Target Genes ◽  
Receptor Gene ◽  
Rna Extraction ◽  
Amplification Efficiency ◽  
Mrna Levels ◽  
Lh Surge ◽  
Total Rna

The aim of this study was to compare the mRNA levels of hormone receptor for progesterone (PR), oestrogen α (ER-α), oestrogen β (ER-β), and oxytocin (OTR) in canine morulae and blastocysts. Ten healthy mature bitches were inseminated based on monitoring vaginal cytology and progesterone concentration. The first insemination was performed on Day 2 after the preovulatory LH surge (progesterone 4 ng mL–1), and the second was performed 48 h later. All females were submitted to ovariohysterectomy (OVH), and the oviduct as well the uterurs were flushed with PBS solution to obtain the embryos. The females were divided into two groups: Group A (n = 5), morulae were collected 8 days after the LH surge and Group B (n = 5), blastocysts were collected 12 days after the LH surge. The pools (n = 10) of embryos (5 embryos/pool) were stored in RNAlater® (Ambion, Life Technologies, USA) at –80°C. The samples were analysed together. The RNA later was removed used PBS calcium free and the total RNA extraction was performed using the Qiagen RNeasy micro-kit (Hildesheim, Germany). Before reverse-transcription (RT) reaction, the total RNA was treated with DNase I Amplification Grade (Invitrogen Life Technologies, Carlsbad, CA, USA). The gene expression of target genes was assessed by real-time RT-qPCR, using SuperScript III for RT and power SYBR Green PCR Master Mix (Applied Biosystems, USA) for cDNA for PCR. The primers for target genes were designed using the software Primer Express® (Applied Biosystems, USA). The gene expression of target genes was normalized by HPRT gene and the relative abundance of mRNA was determined by the ΔΔct method corrected by amplification efficiency using Pffafl’s equation. The means of mRNA relative abundance were compared by t-test. The PR mRNA expression only in blastocysts is similar to the results obtained by Hou et al. (1997) in rat embryos. It is believed that the absence of PR in the early stages of cleavage is due to the indirect action of progesterone by growth factors produced by the maternal reproductive tract (2). Apparently, ER-β action does not occur in the embryo canine phases analysed; however, the action of ER-α seems related to the deployment signal as seen by Hou et al. (1996) in rats. Similarly to findings in the literature, OTR expression decreased in canine embryonic development. This receptor was produced by blastocysts while present in the uterus, which may represent an incidental mechanism to the embryo control of endometrial receptivity, such as also to prevent the development of endometrial luteolytic mechanism. The variation in hormone receptors gene expression in canine embryos can be influencing the transition from morula to blastocyst. In addition, a hormonal influence on these structures can occur in different ways.

scholarly journals NAD-linked mechanisms of gene de-repression and a novel role for CtBP in persistent adenovirus infection of lymphocytes

2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Megan L. Dickherber ◽  
Charlie Garnett-Benson
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Cell Lines ◽  
Lymphocyte Activation ◽  
Viral Gene Expression ◽  
Productive Infection ◽  
Viral Gene ◽  
Global Changes ◽  
Persistently Infected ◽  
Infected Cells

Abstract Background Adenovirus (AdV) infection is ubiquitous in the human population and causes acute infection in the respiratory and gastrointestinal tracts. In addition to lytic infections in epithelial cells, AdV can persist in a latent form in mucosal lymphocytes, and nearly 80% of children contain viral DNA in the lymphocytes of their tonsils and adenoids. Reactivation of latent AdV is thought to be the source of deadly viremia in pediatric transplant patients. Adenovirus latency and reactivation in lymphocytes is not well studied, though immune cell activation has been reported to promote productive infection from latency. Lymphocyte activation induces global changes in cellular gene expression along with robust changes in metabolic state. The ratio of free cytosolic NAD+/NADH can impact gene expression via modulation of transcriptional repressor complexes. The NAD-dependent transcriptional co-repressor C-terminal Binding Protein (CtBP) was discovered 25 years ago due to its high affinity binding to AdV E1A proteins, however, the role of this interaction in the viral life cycle remains unclear. Methods The dynamics of persistently- and lytically-infected cells are evaluated. RT-qPCR is used to evaluate AdV gene expression following lymphocyte activation, treatment with nicotinamide, or disruption of CtBP-E1A binding. Results PMA and ionomycin stimulation shifts the NAD+/NADH ratio in lymphocytic cell lines and upregulates viral gene expression. Direct modulation of NAD+/NADH by nicotinamide treatment also upregulates early and late viral transcripts in persistently-infected cells. We found differential expression of the NAD-dependent CtBP protein homologs between lymphocytes and epithelial cells, and inhibition of CtBP complexes upregulates AdV E1A expression in T lymphocyte cell lines but not in lytically-infected epithelial cells. Conclusions Our data provide novel insight into factors that can regulate AdV infections in activated human lymphocytes and reveal that modulation of cellular NAD+/NADH can de-repress adenovirus gene expression in persistently-infected lymphocytes. In contrast, disrupting the NAD-dependent CtBP repressor complex interaction with PxDLS-containing binding partners paradoxically alters AdV gene expression. Our findings also indicate that CtBP activities on viral gene expression may be distinct from those occurring upon metabolic alterations in cellular NAD+/NADH ratios or those occurring after lymphocyte activation.

scholarly journals Simian Virus 5 Is a Poor Inducer of Chemokine Secretion from Human Lung Epithelial Cells: Identification of Viral Mutants That Activate Interleukin-8 Secretion by Distinct Mechanisms

2003 ◽  
Vol 77 (12) ◽  
pp. 7124-7130 ◽  
Author(s):  
Virginia A. Young ◽  
Griffith D. Parks
Keyword(s):  
Gene Expression ◽  
Human Lung ◽  
A549 Cells ◽  
Viral Gene Expression ◽  
Simian Virus ◽  
Interleukin 8 ◽  
Parainfluenza Virus ◽  
Viral Gene ◽  
Simian Virus 5 ◽  
Infected Cells

ABSTRACT We have compared chemokine secretion from human lung A549 cells infected with simian virus 5 (SV5) with other members of the Rubulavirus genus of paramyxoviruses. High levels of the chemokines interleukin-8 (IL-8) and macrophage chemoattractant protein-1 (MCP-1) were secreted from A549 cells infected with Human parainfluenza virus type 2 (HPIV-2) but not from cells infected with wild-type (WT) SV5. The lack of IL-8 secretion from SV5-infected cells was not due to a global block in all signal transduction pathways leading to IL-8 secretion, since SV5-infected A549 cells secreted IL-8 after stimulation with exogenously added tumor necrosis factor alpha or by coinfection with HPIV-2. A previously described, recombinant SV5 containing substitutions in the shared region of the P/V gene (rSV5-P/V-CPI−) induced IL-8 secretion by a mechanism that was dependent on viral gene expression. By contrast, an SV5 variant isolated from persistently infected cells (Wake Forest strain of Canine parainfluenza virus) induced IL-8 secretion by a mechanism that was largely not affected by inhibitors of viral gene expression. Together, these data demonstrate that SV5 is unusual compared to other closely related paramyxoviruses, since SV5 is a very poor inducer of the cytokines IL-8 and MCP-1. The isolation of two recombinant SV5 mutants that are defective in preventing chemokine induction will allow an identification of mechanisms utilized by WT SV5 to avoid activation of host cell innate immune responses to infection.

Blocking of CDK4 Gene Expression by Antisense Oligos Induce Apoptosis and Decreases the Proliferation of A549 Cells

2012 ◽  
Vol 2 (8) ◽  
pp. 51-53
Author(s):  
Babu R L Babu R L ◽  
◽  
Vijayalakshmi E Vijayalakshmi E ◽  
Naveen Kumar M Naveen Kumar M ◽  
Rajeshwari H. Patil ◽  
...  
Keyword(s):  
Gene Expression ◽  
A549 Cells ◽  
Cdk4 Gene ◽  
Antisense Oligos

scholarly journals Time-resolved transcriptional profiling of Trichinella-infected murine myocytes helps to elucidate host–pathogen interactions in the muscle stage

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Xiaoxiang Hu ◽  
Xiaolei Liu ◽  
Chen Li ◽  
Yulu Zhang ◽  
Chengyao Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Biology ◽  
Trichinella Spiralis ◽  
Expression Patterns ◽  
Muscle Cells ◽  
Host Cells ◽  
Initial Reaction ◽  
Global Changes ◽  
Mammalian Skeletal Muscle ◽  
Time Resolved

Abstract Background Parasites of the genus Trichinella are the pathogenic agents of trichinellosis, which is a widespread and severe foodborne parasitic disease. Trichinella spiralis resides primarily in mammalian skeletal muscle cells. After invading the cells of the host organism, T. spiralis must elude or invalidate the host’s innate and adaptive immune responses to survive. It is necessary to characterize the pathogenesis of trichinellosis to help to prevent the occurrence and further progression of this disease. The aims of this study were to elucidate the mechanisms of nurse cell formation, pathogenesis and immune evasion of T. spiralis, to provide valuable information for further research investigating the basic cell biology of Trichinella-infected muscle cells and the interaction between T. spiralis and its host. Methods We performed transcriptome profiling by RNA sequencing to identify global changes at 1, 3, 7, 10 and 15 days post-infection (dpi) in gene expression in the diaphragm after the parasite entered and persisted within the murine myocytes; the mice were infected by intravenous injection of newborn larvae. Gene expression analysis was based on the alignment results. Differentially expressed genes (DEGs) were identified based on their expression levels in various samples, and functional annotation and enrichment analysis were performed. Results The most extensive and dynamic gene expression responses in host diaphragms were observed during early infection (1 dpi). The number of DEGs and genes annotated in the Kyoto Encyclopedia of Genes and Genomes and Gene Ontology databases decreased significantly in the infected mice compared to the uninfected mice at 3 and 7 dpi, suddenly increased sharply at 10 dpi, and then decreased to a lower level at 15 dpi, similar to that observed at 3 and 7 dpi. The massive initial reaction of the murine muscle cells to Trichinella infection steadied in the later stages of infection, with little additional changes detected for the remaining duration of the studied process. Although there were hundreds of DEGs at each time point, only 11 genes were consistently up- or downregulated at all 5 time points. Conclusions The gene expression patterns identified in this study can be employed to characterize the coordinated response of T. spiralis-infected myocytes in a time-resolved manner. This comprehensive dataset presents a distinct and sensitive picture of the interaction between host and parasite during intracellular infection, which can help to elucidate how pathogens evade host defenses and coordinate the biological functions of host cells to survive in the mammalian environment.

scholarly journals “Limiting access to iron decreases infection of Atlantic salmon SHK-1 cells with bacterium Piscirickettsia salmonis”

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Rodrigo Díaz ◽  
José Troncoso ◽  
Eva Jakob ◽  
Stanko Skugor
Keyword(s):  
Gene Expression ◽  
Iron Deficiency ◽  
Atlantic Salmon ◽  
Bacterial Load ◽  
Host Cells ◽  
Immune Gene ◽  
Immune Signaling ◽  
Immune Effector ◽  
Infected Cells ◽  
Piscirickettsia Salmonis

Abstract Background Vertebrate hosts limit the availability of iron to microbial pathogens in order to nutritionally starve the invaders. The impact of iron deficiency induced by the iron chelator deferoxamine mesylate (DFO) was investigated in Atlantic salmon SHK-1 cells infected with the facultative intracellular bacterium Piscirickettsia salmonis. Results Effects of the DFO treatment and P. salmonis on SHK-1 cells were gaged by assessing cytopathic effects, bacterial load and activity, and gene expression profiles of eight immune biomarkers at 4- and 7-days post infection (dpi) in the control group, groups receiving single treatments (DFO or P. salmonis) and their combination. The chelator appears to be well-tolerated by host cells, while it had a negative impact on the number of bacterial cells and associated cytotoxicity. DFO alone had minor effects on gene expression of SHK-1 cells, including an early activation of IL-1β at 4 dpi. In contrast to few moderate changes induced by single treatments (either infection or chelator), most genes had highest upregulation in the infected groups receiving DFO. The mildest induction of hepcidin-1 (antimicrobial peptide precursor and regulator of iron homeostasis) was observed in cells exposed to DFO alone, followed by P. salmonis infected cells while the addition of DFO to infected cells further increased the mRNA abundance of this gene. Transcripts encoding TNF-α (immune signaling) and iNOS (immune effector) showed sustained increase at both time points in this group while cathelicidin-1 (immune effector) and IL-8 (immune signaling) were upregulated at 7 dpi. The stimulation of protective gene responses seen in infected cultures supplemented with DFO coincided with the reduction of bacterial load and activity (judged by the expression of P. salmonis 16S rRNA), and damage to cultured host cells. Conclusion The absence of immune gene activation under normal iron conditions suggests modulation of host responses by P. salmonis. The negative effect of iron deficiency on bacteria likely allowed host cells to respond in a more protective manner to the infection, further decreasing its progression. Presented findings encourage in vivo exploration of iron chelators as a promising strategy against piscirickettsiosis.

scholarly journals Metatranscriptomic analysis of colonic microbiota’s functional response to different dietary fibers in growing pigs

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Jie Xu ◽  
Rongying Xu ◽  
Menglan Jia ◽  
Yong Su ◽  
Weiyun Zhu
Keyword(s):  
Gene Expression ◽  
Functional Response ◽  
Relative Abundance ◽  
Expression Profiles ◽  
Nutrient Digestibility ◽  
Growing Pigs ◽  
Control Group ◽  
Dietary Fibers ◽  
Microbial Composition ◽  
Colonic Microbiota

Abstract Background Dietary fibers are widely considered to be beneficial to health as they produce nutrients through gut microbial fermentation while facilitating weight management and boosting gut health. To date, the gene expression profiles of the carbohydrate active enzymes (CAZymes) that respond to different types of fibers (raw potato starch, RPS; inulin, INU; pectin, PEC) in the gut microbes of pigs are not well understood. Therefore, we investigated the functional response of colonic microbiota to different dietary fibers in pigs through metatranscriptomic analysis. Results The results showed that the microbial composition and CAZyme structure of the three experimental groups changed significantly compared with the control group (CON). Based on a comparative analysis with the control diet, RPS increased the abundance of Parabacteroides, Ruminococcus, Faecalibacterium and Alloprevotella but decreased Sutterella; INU increased the relative abundance of Fusobacterium and Rhodococcus but decreased Bacillus; and PEC increased the relative abundance of the Streptococcus and Bacteroidetes groups but decreased Clostridium, Clostridioides, Intestinibacter, Gemmiger, Muribaculum and Vibrio. The gene expression of CAZymes GH8, GH14, GH24, GH38, GT14, GT31, GT77 and GT91 downregulated but that of GH77, GH97, GT3, GT10 and GT27 upregulated in the RPS diet group; the gene expression of AA4, AA7, GH14, GH15, GH24, GH26, GH27, GH38, GH101, GT26, GT27 and GT38 downregulated in the INU group; and the gene expression of PL4, AA1, GT32, GH18, GH37, GH101 and GH112 downregulated but that of CE14, AA3, AA12, GH5, GH102 and GH103 upregulated in the PEC group. Compared with the RPS and INU groups, the composition of colonic microbiota in the PEC group exhibited more diverse changes with the variation of CAZymes and Streptococcus as the main contributor to CBM61, which greatly promoted the digestion of pectin. Conclusion The results of this exploratory study provided a comprehensive overview of the effects of different fibers on nutrient digestibility, gut microbiota and CAZymes in pig colon, which will furnish new insights into the impacts of the use of dietary fibers on animal and human health.

scholarly journals Interaction of 7SK with the Smn complex modulates snRNP production

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Changhe Ji ◽  
Jakob Bader ◽  
Pradhipa Ramanathan ◽  
Luisa Hennlein ◽  
Felix Meissner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptional Regulation ◽  
Fine Tuning ◽  
Global Changes ◽  
Functional Association ◽  
Transcriptional Inhibition ◽  
Smn Complex ◽  
Core Components

AbstractGene expression requires tight coordination of the molecular machineries that mediate transcription and splicing. While the interplay between transcription kinetics and spliceosome fidelity has been investigated before, less is known about mechanisms regulating the assembly of the spliceosomal machinery in response to transcription changes. Here, we report an association of the Smn complex, which mediates spliceosomal snRNP biogenesis, with the 7SK complex involved in transcriptional regulation. We found that Smn interacts with the 7SK core components Larp7 and Mepce and specifically associates with 7SK subcomplexes containing hnRNP R. The association between Smn and 7SK complexes is enhanced upon transcriptional inhibition leading to reduced production of snRNPs. Taken together, our findings reveal a functional association of Smn and 7SK complexes that is governed by global changes in transcription. Thus, in addition to its canonical nuclear role in transcriptional regulation, 7SK has cytosolic functions in fine-tuning spliceosome production according to transcriptional demand.

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