scholarly journals The Transcriptional landscape of Streptococcus pneumoniae reveals a complex operon architecture and abundant riboregulation critical for growth and virulence

2018 ◽  
Author(s):  
Indu Warrier ◽  
Nikhil Ram-Mohan ◽  
Zeyu Zhu ◽  
Ariana Hazery ◽  
Michelle M Meyer ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
High Throughput ◽  
Transcription Initiation ◽  
High Throughput Sequencing ◽  
Regulatory Element ◽  
Fine Tuning ◽  
Transcriptional Units ◽  
Rna Elements ◽  
Transcriptional Landscape

AbstractEfficient and highly organized transcription initiation and termination is fundamental to an organism’s ability to survive, proliferate, and quickly respond to its environment. Over the last decade, our simplistic outlook of bacterial transcriptional regulation and architecture has evolved to include stimulus-responsive regulation by untranslated RNA and the formation of alternate transcriptional units. In this study, we map the transcriptional landscape of the bacterial pathogen Streptococcus pneumoniae by applying a combination of high-throughput RNA-sequencing techniques. Our study reveals a complex transcriptome wherein environment-respondent alternate transcriptional units are observed within operons stemming from internal transcription start sites (TSS) and transcription terminators (TTS) suggesting that more fine-tuning of regulation occurs than previously thought. Additionally, we identify many putative cis-regulatory RNA elements and riboswitches within 5’-untranslated regions (5’-UTR) of genes. By integrating TSSs and TTSs with independently collected RNA-Seq datasets from a variety of conditions, we establish the response of these regulators to changes in growth conditions and validate several of them. Furthermore, to determine the importance of ribo-regulation by 5’-UTR elements for in vivo virulence, we show that the pyrR regulatory element is essential for survival, successful colonization and infection in mice suggesting that such RNA elements are potential drug targets. Importantly, we show that our approach of combining high-throughput sequencing with in vivo experiments can reconstruct a global understanding of regulation, but also pave the way for discovery of compounds that target (ribo-) regulators to mitigate virulence and antibiotic resistance.

scholarly journals CDK10 Regulates Gastric Cancer Metastasis By Inhibiting lncRNA-C5ORF42-5 Via Phosphorylation Level of AKT/ERK

2021 ◽  
Author(s):  
Zi-Jian Deng ◽  
Dong-Wen Chen ◽  
Xi-Jie Chen ◽  
Jia-Ming Fang ◽  
Liang Xv ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cancer Cells ◽  
Cell Line ◽  
High Throughput ◽  
Cancer Metastasis ◽  
High Throughput Sequencing ◽  
Gastric Cancer Cells ◽  
Related Proteins

Abstract Background: Gastric cancer is the fourth most common malignant disease. Both CDK10 and long noncoding RNAs (lncRNAs) have been found to exert biological functions in multiple cancers. However, it is still unclear whether CDK10 represses tumor progression in gastric cancer by reducing potential targeting lncRNAs.Methods: The functions of CDK10 and lncRNA-C5ORF42-5 in proliferation, invasion and migration were assessed by MTS assays, colony formation assays, cell cycle and apoptosis assays, Transwell assays, wound healing assays and animal experiments. We used high-throughput sequencing to confirm the existence of lncRNA-C5ORF42-5 and quantitative real-time PCR was used to evaluate lncRNA expression. Then, with RNA-seq sequencing as well as GO function and KEGG enrichment analysis, we identified the signaling pathways in which lncRNA-C5ORF42-5 was involved in gastric cancer. Finally, western blotting was used to identify the genes regulated by lncRNA-C5ORF42-5.Results: Our results showed that CDK10 is expressed at relatively low levels in gastric cancer cell lines and inhibits the progression of gastric cancer cells both in vitro and in vivo. Next, based on high-throughput sequencing, we identified a novel lncRNA, lncRNA-C5ORF42-5, in the stable CDK10-overexpressing cell line compared with the CDK-knockdown cell line and their controls. Additionally, we confirmed that lncRNA-C5ORF42-5 acts as an oncogene to promote metastasis in gastric cancer in vitro and in vivo. We then ascertained that lncRNA-C5ORF42-5 is a major contributor to the function of CDK10 in gastric cancer metastasis by upregulating lncRNA-C5ORF42-5 to reverse the effects of CDK10 overexpression. Finally, we explored the mechanism by which lncRNA-C5ORF42-5 overexpression affects gastric cancer cells to elucidate whether lncRNA-C5ORF42-5 may increase the activity of the SMAD pathway of BMP signaling and promote the expression of EMT-related proteins, such as E-cadherin. Additionally, overexpression of lncRNA-C5ORF42-5 affected the phosphorylation levels of AKT and ERK.Conclusion: Our findings suggest that CDK10 overexpression represses gastric cancer tumor progression by reducing lncRNA-C5ORF42-5 and hindering activation of the related proteins in metastatic signaling pathways, which provides new insight into developing effective therapeutic strategies in the treatment of metastatic gastric cancer.

scholarly journals T cell neoepitope discovery in colorectal cancer by high throughput profiling of somatic mutations in expressed genes

Gut ◽  
2015 ◽  
Vol 66 (3) ◽  
pp. 454-463 ◽  
Author(s):  
Daniele Mennonna ◽  
Cristina Maccalli ◽  
Michele C Romano ◽  
Claudio Garavaglia ◽  
Filippo Capocefalo ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Cell Epitope ◽  
Patient Specific ◽  
Cancer Genes ◽  
Healthy Donors

ObjectivePatient-specific (unique) tumour antigens, encoded by somatically mutated cancer genes, generate neoepitopes that are implicated in the induction of tumour-controlling T cell responses. Recent advancements in massive DNA sequencing combined with robust T cell epitope predictions have allowed their systematic identification in several malignancies.DesignWe undertook the identification of unique neoepitopes in colorectal cancers (CRCs) by using high-throughput sequencing of cDNAs expressed by standard cancer cell cultures, and by related cancer stem/initiating cells (CSCs) cultures, coupled with a reverse immunology approach not requiring human leukocyte antigen (HLA) allele-specific epitope predictions.ResultsSeveral unique mutated antigens of CRC, shared by standard cancer and related CSC cultures, were identified by this strategy. CD8+and CD4+T cells, either autologous to the patient or derived from HLA-matched healthy donors, were readily expanded in vitro by peptides spanning different cancer mutations and specifically recognised differentiated cancer cells and CSC cultures, expressing the mutations. Neoepitope-specific CD8+T cell frequency was also increased in a patient, compared with healthy donors, supporting the occurrence of clonal expansion in vivo.ConclusionsThese results provide a proof-of-concept approach for the identification of unique neoepitopes that are immunogenic in patients with CRC and can also target T cells against the most aggressive CSC component.

scholarly journals Molecular Fitness Landscapes from High-Coverage Sequence Profiling

2019 ◽  
Vol 48 (1) ◽  
pp. 1-18 ◽  
Author(s):  
Celia Blanco ◽  
Evan Janzen ◽  
Abe Pressman ◽  
Ranajay Saha ◽  
Irene A. Chen
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Fitness Landscape ◽  
Complete Sequence ◽  
Fitness Landscapes ◽  
Future Research ◽  
High Coverage

The function of fitness (or molecular activity) in the space of all possible sequences is known as the fitness landscape. Evolution is a random walk on the fitness landscape, with a bias toward climbing hills. Mapping the topography of real fitness landscapes is fundamental to understanding evolution, but previous efforts were hampered by the difficulty of obtaining large, quantitative data sets. The accessibility of high-throughput sequencing (HTS) has transformed this study, enabling large-scale enumeration of fitness for many mutants and even complete sequence spaces in some cases. We review the progress of high-throughput studies in mapping molecular fitness landscapes, both in vitro and in vivo, as well as opportunities for future research. Such studies are rapidly growing in number. HTS is expected to have a profound effect on the understanding of real molecular fitness landscapes.

scholarly journals Transcription of Drosophila Troponin I Gene Is Regulated by Two Conserved, Functionally Identical, Synergistic Elements

2004 ◽  
Vol 15 (3) ◽  
pp. 1185-1196 ◽  
Author(s):  
María-Cruz Marín ◽  
José-Rodrigo Rodríguez ◽  
Alberto Ferrús
Keyword(s):  
Transcription Initiation ◽  
Troponin I ◽  
Regulatory Element ◽  
Expression Patterns ◽  
In Silico Analysis ◽  
Initiation Site ◽  
Transcription Initiation Site ◽  
Upstream Regulatory Element ◽  
Transgenic Lines

The Drosophila wings-up A gene encodes Troponin I. Two regions, located upstream of the transcription initiation site (upstream regulatory element) and in the first intron (intron regulatory element), regulate gene expression in specific developmental and muscle type domains. Based on LacZ reporter expression in transgenic lines, upstream regulatory element and intron regulatory element yield identical expression patterns. Both elements are required for full expression levels in vivo as indicated by quantitative reverse transcription-polymerase chain reaction assays. Three myocyte enhancer factor-2 binding sites have been functionally characterized in each regulatory element. Using exon specific probes, we show that transvection is based on transcriptional changes in the homologous chromosome and that Zeste and Suppressor of Zeste 3 gene products act as repressors for wings-up A. Critical regions for transvection and for Zeste effects are defined near the transcription initiation site. After in silico analysis in insects (Anopheles and Drosophila pseudoobscura) and vertebrates (Ratus and Coturnix), the regulatory organization of Drosophila seems to be conserved. Troponin I (TnI) is expressed before muscle progenitors begin to fuse, and sarcomere morphogenesis is affected by TnI depletion as Z discs fail to form, revealing a novel developmental role for the protein or its transcripts. Also, abnormal stoichiometry among TnI isoforms, rather than their absolute levels, seems to cause the functional muscle defects.

scholarly journals Characterization of Intracellular Growth RegulatoricgRby Utilizing Transcriptomics To Identify Mediators of Pathogenesis in Shigella flexneri

2013 ◽  
Vol 81 (9) ◽  
pp. 3068-3076 ◽  
Author(s):  
Carolyn R. Morris ◽  
Christen L. Grassel ◽  
Julia C. Redman ◽  
Jason W. Sahl ◽  
Eileen M. Barry ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Diarrheal Disease ◽  
Intracellular Growth ◽  
Bioinformatics Analyses ◽  
Content Type ◽  
Regulatory Pathways

ABSTRACTShigellaspecies Gram-negative bacteria which cause a diarrheal disease, known as shigellosis, by invading and destroying the colonic mucosa and inducing a robust inflammatory response. With no vaccine available, shigellosis annually kills over 600,000 children in developing countries. This study demonstrates the utility of combining high-throughput bioinformatic methods within vitroandin vivoassays to provide new insights into pathogenesis. Comparisons ofin vivoandin vitrogene expression identified genes associated with intracellular growth. Additional bioinformatics analyses identified genes that are present inS. flexneriisolates but not in the three otherShigellaspecies. Comparison of these two analyses revealed nine genes that are differentially expressed during invasion and that are specific toS. flexneri. One gene, a DeoR family transcriptional regulator with decreased expression during invasion, was further characterized and is now designatedicgR, forintracellulargrowthregulator. Deletion oficgRcaused no difference in growthin vitrobut resulted in increased intracellular replication in HCT-8 cells. Furtherin vitroandin vivostudies using high-throughput sequencing of RNA transcripts (RNA-seq) of an isogenic ΔicgRmutant identified 34 genes that were upregulated under both growth conditions. This combined informatics and functional approach has allowed the characterization of a gene and pathway previously unknown inShigellapathogenesis and provides a framework for further identification of novel virulence factors and regulatory pathways.

Regulation of the PU.1 Gene by Sense and Functional Antisense RNAs Generated through the Same Chromatin Architecture.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 781-781
Author(s):  
Alex Ebralidze ◽  
Pu Zhang ◽  
Frank Rosenbauer ◽  
Gang Huang ◽  
Ulrich Steidl ◽  
...  
Keyword(s):  
Regulatory Element ◽  
Cell Types ◽  
Antisense Transcription ◽  
Fine Tuning ◽  
Nuclear Fraction ◽  
Antisense Transcripts ◽  
Chromosome Conformation ◽  
Antisense Rnas ◽  
Chromatin Architecture

Abstract The transcription factor PU.1 is an important regulator of hematopoiesis and correct expression levels in specific lineages are critical for normal hematopoietic development. Specifically, PU.1 is maintained or upregulated in specific lineages, and failure to downregulate PU.1 in other lineages can lead to a block in development of that lineage and/or leukemia. In vivo expression of PU.1 is dependent on an upstream regulatory element called the URE. Disruption of the URE leads to downregulation of PU.1 and development of leukemia and lymphoma, but the other distal elements regulating PU.1 have not been defined. Here we show that other phylogenetically conserved elements participate in the initiation of antisense transcription, and that these antisense RNAs function as important modulators of proper dosages of PU.1. Specifically, antisense transcripts originate from specific conserved sites in introns 1 and 3, and that the intron 3 site contains binding sites for transcription factors such as AML1 and Ets factors. The conserved intron 3 element also possesses anti-sense promoter activity. These antisense transcripts are present at about 15% of PU.1 sense transcripts in PU.1 expressing cells. They negatively regulate PU.1 sense RNA, as introduction of siRNA molecules which specifically target antisense transcripts lead to 3–5 fold increases in PU.1 sense RNA and protein. Both sense and antisense PU.1 gene RNAs are dependent on the URE and are transcribed from the same chromatin architecture, in which the conserved elements, including URE and sense and antisense promoters are located in the same nuclear fraction and can be shown to exist in the same nucleoprotein complex by chromosome conformation capture (3C). We are currently testing the mechanisms involved in the formation of such complexes, and specifically whether the complexes are mediated by binding of AML1 to the URE. Since we do not observe significant differences in antisense transcript levels between PU.1 high-expressing and PU.1 low-expressing cells, we hypothesize that the function of these antisense transcripts is to modulate rather than absolutely control PU.1 levels: in PU.1 high-expressing cells, such as myeloid cells, the antisense transcripts trim PU.1 levels to prevent overexpression, while in cell types in which PU.1 is not expressed, such as T cells, the antisense transcripts prevent any expression of PU.1. We propose that such a mechanism will likely be important in fine-tuning the regulation of many genes and may be the reason for the large number of overlapping complementary transcripts with so far unknown function.

The Novel FLT3-N676K Mutant Induces Acute Leukemia Independently of the Inv(16) Chimeric Gene CBFB-MYH11

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1383-1383
Author(s):  
Kezhi Huang ◽  
Min Yang ◽  
Zengkai Pan ◽  
Florian H. Heidel ◽  
Michaela Scherr ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
De Novo ◽  
Chimeric Gene ◽  
Leukemic Cells ◽  
Single Amino Acid ◽  
Hematopoietic Stem ◽  
Transforming Activity

Abstract Using high-throughput sequencing, an increased number of gene mutations has been identified in cancer. Among the up to hundreds of acquired mutations in cancer clones, only a few cooperating mutations are believed to be needed for initiation of the malignant disease. Recently, we reported a single amino acid substitution at position 676 (N676K) within the FLT3 kinase domain as the sole cause of resistance to PKC412 in one patient with FLT3-ITD associated acute myeloid leukemia (AML). The FLT3-N676K mutation was more recently identified independently in up to 6% of de novo AML patients with inv(16) by other groups. As FLT3-TKD mutations are strongly associated with inv(16) in AML and particularly FLT3-N676K was found almost exclusively in AML patients with inv(16), this prompted us to investigate the transforming activity of FLT3-N676K and to test whether FLT3-N676K would cooperate with inv(16) to promote AML. First, we analyzed in vivo leukemogenesis mediated by FLT3-N676K. Retroviral expression of FLT3-N676K in myeloid 32D cells induced AML in syngeneic C3H/HeJ mice (n=11/13, latency ~8 weeks), with a transforming activity similar to FLT3-ITD (n=8/8), FLT3-TKD D835Y (n=8/9), and FLT3-ITD-N676K (n=9/9) mutations. Three out of 14 C57BL/6J mice transplanted with FLT3-N676K-transduced primary lineage negative (Lin-) bone marrow cells died of acute leukemia (latency of 68, 77, and 273 days), while none of 16 animals in the control groups including FLT3-ITD and CBFß-SMMHC developed any hematological malignancy. Secondly, co-expression of FLT3-N676K and CBFß-SMMHC did not promote acute leukemia in 3 independent experiments using C3H/HeJ and C57BL/6J mice (n=16). So far only 1 out of 11 C57BL/6J mice co-expressing FLT3-N676K and CBFß-SMMHC developed acute leukemia (AML with latency of 166 days). In comparison with FLT3-ITD, FLT3-N676K tended to result in stronger phosphorylation of FLT3, MAPK and AKT, and diseased animals carrying FLT3-N676K demonstrated much lower frequency of leukemic stem cells in the majority of analyzed cases. Importantly, leukemic cells co-expressing FLT3-N676K and CBFß-SMMHC were still highly sensitive to the FLT3 inhibitor AC220. Taken together, we show that FLT3-N676K mutant is potent to transform murine hematopoietic stem/progenitor cells in vivo independently of the inv(16) chimeric gene CBFB-MYH11. This is the first report of acute leukemia induced by an activating FLT3 mutation in C57BL/6J mice. Moreover, our data suggest that targeting FLT3-N676K mutation may be an attractive treatment option for FLT3-N676K-positive patients without concurrent ITD. Our data emphasize more careful analysis of the cooperating network of mutations identified in AML by high-throughput sequencing. This work was supported by DJCLS (grant: 13/22) and the Deutsche Forschungsgemeinschaft (grant: Li 1608/2-1). KH and ZP were supported by the China Scholarship Council (2011638024 and 201406100008). Disclosures Heidel: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Antigenic and genetic characterization of Streptococcus pneumoniae strains isolated from patients with invasive and non-invasive pneumococcal infections by using high-throughput sequencing

2021 ◽  
Vol 98 (5) ◽  
pp. 512-518
Author(s):  
K. O. Mironov ◽  
I. I. Gaponova ◽  
V. I. Korchagin ◽  
Yu. V. Mikhailova ◽  
A. A. Shelenkov ◽  
...  
Keyword(s):  
Data Analysis ◽  
Streptococcus Pneumoniae ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Whole Genome ◽  
Pneumococcal Infections ◽  
Genome Sequences ◽  
Illumina Platform ◽  
Genetic Characteristics ◽  
Non Invasive

The objective of this study was to characterize and compare antigenic and genetic characteristics of Streptococcus pneumoniae strains isolated from patients with invasive and non-invasive pneumococcal infections (PIs) by using the data of high-throughput sequencing.Materials and methods. A total of 158 S. pneumoniae strains were studied. All of them were isolated during different stages of the PEHASus multicenter study performed in 2015-2020. The data analysis was based on the information about whole-genome sequences of 46 strains isolated during the above study. Real-time PCR methods and high-throughput sequencing (the Illumina platform) were used for identification of serotypes. The SeroBA, PneumoCaT software and PubMLST.org website resources were used in the data processing.Results and discussion. The serotypes of all the studied strains were identified. A number of discrepancies among serotypes in serogroup 6 and one discordant result were revealed by the analysis of whole-genome sequences using 2 programs. The PCR methods were effectively used to characterize serotypes in 87% and 69% of the pathogens of invasive and non-invasive PIs, respectively. The serotypes contained in PCV13 accounted for 59% and 37%, while PPV23 serotypes accounted for 78% and 53% of the strains isolated from patients with invasive and non-invasive PIs, respectively. The data analysis was unable to identify either the dominant sequence type (a total of 81 sequence types have been identified) or clonal complexes, except for serotype 3 strains, thus demonstrating consistency with the data from previous studies suggesting the absence of a well-represented clonal structure of S. pneumoniae associated with pneumococcal meningitis in Russia.Conclusion. The obtained data made it possible to identify the distribution of the circulating serotypes and genetic characteristics of the strains isolated from PI patients, thus being instrumental for assessment of the effectiveness of the existing polyvalent vaccines and providing information for improvement of the PCR-based methods of serotyping.

scholarly journals Identification of Drug Targets and Potential Molecular Mechanisms of Wantong Jingu Tablet in Treatment of Rats with Collagen-Induced Arthritis based on 16S rDNA High-Throughput Sequencing and Metabolomic Analysis

2020 ◽  
Author(s):  
Zhaodong Li ◽  
Fangyuan Qi ◽  
Fan Li
Keyword(s):  
16S Rdna ◽  
High Throughput ◽  
Molecular Mechanisms ◽  
High Throughput Sequencing ◽  
Caspase 3 ◽  
Collagen Induced Arthritis ◽  
Apoptosis Pathway ◽  
Bacterial Phyla ◽  
Joint Pathology

Abstract Background: Wantong Jingu Tablet (WJT), a mixture of traditional Chinese medicine, can reduce the symptoms of rheumatoid arthritis (RA), but its pharmacological mechanism is unclear. The aims of this study were to investigate the therapeutic mechanisms of WJT for RA in vivo.Methods: The effects of WJT on the joint pathology, and the levels of Bax, Bcl-2,caspase-3, cleaved-caspase-3, ERK1/2, pERK1/2, TNF-α, IL-1β, and IL-6 were demonstrated based on several experiments in the model of collagen-induced arthritis (CIA) in rats. 16S rDNA high-throughput sequencing was used to investigate the effect of WJT on the overall structure and composition of gut microbiota. Meanwhile, metabolite changes in faeces were analyzed by metabolomics techniques. Results: The results showed that WJT restored the joint pathology in CIA rats, upregulated Bax and cleaved-caspase-3, downregulated Bcl-2, caspase-3, and pERK1/2, and reduced the levels of pro-inflammatory cytokines. The overall gut microbial structure in CIA rats was altered after WJT treatment. Three bacterial phyla were prominently restored: Bacteroidetes,Tenericutes and Deferribacteres, and three bacterial genera were significantly reversed: Vibrio, Macrococcus and Vagococcus. Furthermore, five specific metabolites associated with these specific bacterial genera were identified by correlation analysis. In addition, WJT supplement trended to down-regulate the other five metabolites according to metabolomic analyses. Conclusions: These results revealed that WJT restored the pathological changes of RA might through activating the mitochondrial apoptosis pathway, inhibited MEK/ERK signaling, and modulating the special bacteria and the special metabolites.

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