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 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 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.

scholarly journals High-throughput inverse toeprinting reveals the complete sequence dependence of ribosome-targeting antibiotics

2018 ◽  
Author(s):  
Britta Seip ◽  
Guénaёl Sacheau ◽  
Denis Dupuy ◽  
C. Axel Innis
Keyword(s):  
High Throughput ◽  
Quantitative Measure ◽  
Complete Sequence ◽  
Ribosome Profiling ◽  
Sequence Specificity ◽  
Messenger Rnas ◽  
Coding Region ◽  
Sequence Dependence

It has recently become clear that various antibiotics block the translation of bacterial proteins in a sequence-specific manner. In order to understand how this specificity contributes to antibiotic potency and select better antimicrobial leads, new high-throughput tools are needed. Here, we present inverse toeprinting, a new method to map the position of ribosomes arrested on messenger RNAs during in vitro translation. Unlike ribosome profiling, our method protects the entire coding region upstream of a stalled ribosome, making it possible to work with transcript libraries that randomly sample the sequence space. We used inverse toeprinting to characterize the pausing landscape of free and drug-bound bacterial ribosomes engaged in translation. We obtained a comprehensive list of arrest motifs that could be validated in vivo, along with a quantitative measure of their pause strength. Thus, our method provides a highly parallel and scalable means to characterize the sequence specificity of translation inhibitors.

scholarly journals A Comprehensive Analysis of In Vitro and In Vivo Genetic Fitness of Pseudomonas aeruginosa Using High-Throughput Sequencing of Transposon Libraries

2013 ◽  
Vol 9 (9) ◽  
pp. e1003582 ◽  
Author(s):  
David Skurnik ◽  
Damien Roux ◽  
Hugues Aschard ◽  
Vincent Cattoir ◽  
Deborah Yoder-Himes ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Comprehensive Analysis ◽  
Genetic Fitness

scholarly journals CircRNA_0000392 Promotes Colorectal Cancer Progression by Sponging miR-193a-5p

2020 ◽  
Author(s):  
Hanchen Xu ◽  
Yujing Liu ◽  
Peiqiu Cheng ◽  
Chunyan Wang ◽  
Yang Liu ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Expression Profile ◽  
High Throughput ◽  
Molecular Mechanisms ◽  
High Throughput Sequencing ◽  
Malignant Progression ◽  
Luciferase Assay ◽  
Circular Rnas

Abstract Background: Circular RNAs (circRNAs), an important member of the non-coding RNA family, have been revealed the role in the pathogenic progression of diseases in recent years, particularly in the malignant progression of cancer. With the application of high-throughput sequencing technology, a large number of circRNAs have been found in tumor tissues, and some circRNAs have demonstrated the role as oncogenic genes. In this study, we analyzed the circRNA expression profile in colorectal cancer (CRC) tissues and normal adjacent tissues by high-throughput sequencing, focusing on the circRNA_0000392, a circRNA with significantly increased expression in colorectal cancer tissues, and further investigating its function in the progression of colorectal cancer.Methods: The expression profile of circRNAs in 6 pairs of CRC tissues and normal adjacent tissues was analyzed by RNA-sequencing. We verified the differential circRNAs with expanded samples by qRT-PCR, focused on circRNA_0000392, and evaluated its associations with clinicopathological features. Then we knocked down circRNA_0000392 in CRC cells and evaluated the effect in vitro and in vivo by functional experiments. The dual luciferase assay and RNA pull-down were performed to further explore the downstream potential molecular mechanisms.Results: CircRNA_0000392 was significantly up-regulated in CRC compared with normal adjacent tissues and cell line. The expression level of circRNA_0000392 was positively correlated with the malignant progression of CRC. Functional studies revealed that reducing the expression of circRNA_0000392 could inhibit the proliferation and invasion of CRC both in vitro and in vivo. Mechanistically, circRNA­_0000392 could act as a sponge of miR-193a-5p and regulate the expression of PIK3R3, then affect the activation of the AKT-mTOR pathway in CRC cells.Conclusions: The circRNA_0000392 has the function as an oncogene through miR-193a-5p/PIK3R3-Akt axis in CRC cells, implying that circRNA_0000392 is a potential therapeutic target for the treatment of colorectal cancer and a predictive marker for CRC patients.

scholarly journals Melanogenesis Using Tyrosinate (Not Tyrosinase)

2018 ◽  
Author(s):  
Koen Vercruysse ◽  
Nahfisa Richardson
Keyword(s):  
Large Scale ◽  
Culture Media ◽  
Disodium Salt ◽  
Size Exclusion ◽  
Future Research ◽  
Vis Spectroscopy ◽  
Set Up ◽  
Mediated Oxidation

<p>We present our initial observations regarding the effect of the presence of L-tyrosinate (= L-tyrosine disodium salt) on the auto- or Fe<sup>2+</sup>/H<sub>2</sub>O<sub>2</sub>-mediated oxidation of various catecholic substances into melanin-like pigments. We observed that L-tyrosinate inhibited the Fe<sup>2+</sup>/H<sub>2</sub>O<sub>2</sub>-mediated oxidation. In contrast, L-tyrosinate promoted the auto-oxidation of ortho-diphenols like L-DOPA, dopamine, epinephrine, norepinephrine, catechol or pyrogallol, but not a meta-diphenol like resorcinol. In addition, we briefly demonstrated the melanogenic properties of cell culture media containing L-tyrosinate. The reactions were monitored using UV-Vis spectroscopy and size exclusion chromatography. For a reaction between L-tyrosinate and L-DOPA, a large scale experiment was set up allowing us to isolate, purify and characterize using FT-IR spectroscopy the melanin-like material obtained. We discuss our observations in the context of the <i>in vitro</i> and <i>in vivo</i> study of melanogenesis and provide some directions for future research efforts.<i></i></p>

scholarly journals Melanogenesis Using Tyrosinate (Not Tyrosinase)

2018 ◽  
Author(s):  
Koen Vercruysse ◽  
Nahfisa Richardson
Keyword(s):  
Large Scale ◽  
Culture Media ◽  
Disodium Salt ◽  
Size Exclusion ◽  
Future Research ◽  
Vis Spectroscopy ◽  
Set Up ◽  
Mediated Oxidation

<p>We present our initial observations regarding the effect of the presence of L-tyrosinate (= L-tyrosine disodium salt) on the auto- or Fe<sup>2+</sup>/H<sub>2</sub>O<sub>2</sub>-mediated oxidation of various catecholic substances into melanin-like pigments. We observed that L-tyrosinate inhibited the Fe<sup>2+</sup>/H<sub>2</sub>O<sub>2</sub>-mediated oxidation. In contrast, L-tyrosinate promoted the auto-oxidation of ortho-diphenols like L-DOPA, dopamine, epinephrine, norepinephrine, catechol or pyrogallol, but not a meta-diphenol like resorcinol. In addition, we briefly demonstrated the melanogenic properties of cell culture media containing L-tyrosinate. The reactions were monitored using UV-Vis spectroscopy and size exclusion chromatography. For a reaction between L-tyrosinate and L-DOPA, a large scale experiment was set up allowing us to isolate, purify and characterize using FT-IR spectroscopy the melanin-like material obtained. We discuss our observations in the context of the <i>in vitro</i> and <i>in vivo</i> study of melanogenesis and provide some directions for future research efforts.<i></i></p>

scholarly journals CircRNA_0000392 promotes colorectal cancer progression through the miR-193a-5p/PIK3R3/AKT axis

2020 ◽  
Author(s):  
Hanchen Xu ◽  
Yujing Liu ◽  
Peiqiu Cheng ◽  
Chunyan Wang ◽  
Yang Liu ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Expression Profile ◽  
High Throughput ◽  
Noncoding Rna ◽  
Molecular Mechanisms ◽  
High Throughput Sequencing ◽  
Malignant Progression ◽  
Circular Rnas

Abstract Background: Circular RNAs (circRNAs), important members of the noncoding RNA family, have been recently revealed to play a role in the pathogenic progression of diseases, particularly in the malignant progression of cancer. With the application of high-throughput sequencing technology, a large number of circRNAs have been identified in tumor tissues, and some circRNAs have been demonstrated to act as oncogenes. In this study, we analyzed the circRNA expression profile in colorectal cancer (CRC) tissues and normal adjacent tissues by high-throughput sequencing. We focused on circRNA_0000392, a circRNA with significantly increased expression in CRCtissues, and further investigated its function in the progression of colorectal cancer.Methods: The expression profile of circRNAs in 6 pairs of CRC tissues and normal adjacent tissues was analyzed by RNA sequencing. We verified the identified differentially expressed circRNAs in additional samples by qRT-PCR and selected circRNA_0000392 to evaluate its associations with clinicopathological features. Then, we knocked down circRNA_0000392 in CRC cells and investigated the in vitro and in vivo effects using functional experiments. Dual luciferase and RNA pull-down assays were performed to further explore the downstream potential molecular mechanisms.Results: CircRNA_0000392 was significantly upregulated in CRC compared with normal adjacent tissues and cell lines. The expression level of circRNA_0000392 was positively correlated with the malignant progression of CRC. Functional studies revealed that reducing the expression of circRNA_0000392 could inhibit the proliferation and invasion of CRC both in vitro and in vivo. Mechanistically, circRNA­_0000392 could act as a sponge of miR-193a-5p and regulate the expression of PIK3R3, affecting the activation of the AKT-mTOR pathway in CRC cells.Conclusions: CircRNA_0000392 functions as an oncogene through the miR-193a-5p/PIK3R3/Akt axis in CRC cells, suggesting that circRNA_0000392 is a potential therapeutic target for the treatment of colorectal cancer and a predictive marker for CRC patients.

scholarly journals LABRADOR—A Computational Workflow for Virus Detection in High-Throughput Sequencing Data

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2541
Author(s):  
Izabela Fabiańska ◽  
Stefan Borutzki ◽  
Benjamin Richter ◽  
Hon Q. Tran ◽  
Andreas Neubert ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
De Novo ◽  
Virus Detection ◽  
Third Party ◽  
Reference Sequence ◽  
Clinical Samples ◽  
Sequencing Data

High-throughput sequencing (HTS) allows detection of known and unknown viruses in samples of broad origin. This makes HTS a perfect technology to determine whether or not the biological products, such as vaccines are free from the adventitious agents, which could support or replace extensive testing using various in vitro and in vivo assays. Due to bioinformatics complexities, there is a need for standardized and reliable methods to manage HTS generated data in this field. Thus, we developed LABRADOR—an analysis pipeline for adventitious virus detection. The pipeline consists of several third-party programs and is divided into two major parts: (i) direct reads classification based on the comparison of characteristic profiles between reads and sequences deposited in the database supported with alignment of to the best matching reference sequence and (ii) de novo assembly of contigs and their classification on nucleotide and amino acid levels. To meet the requirements published in guidelines for biologicals’ safety we generated a custom nucleotide database with viral sequences. We tested our pipeline on publicly available HTS datasets and showed that LABRADOR can reliably detect viruses in mixtures of model viruses, vaccines and clinical samples.

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